Generation and utilization of synthetic combinatorial libraries

The use of combinatorial chemistry is fundamentally changing the pace and scope of basic research and drug discovery. Since the introduction of synthetic peptide libraries several years ago, combinatorial chemistry has proven to be a powerful tool for the generation of immense molecular diversities of peptides, peptidomimetics and new organic compounds. This article briefly reviews methods for the generation and application of combinatorial libraries, with particular emphasis on soluble synthetic combinatorial libraries. The utility of these molecular diversities for basic research and drug discovery has been demonstrated through the identification of numerous highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.     

A retrospective analysis of 1570 appendiceal carcinoids

The design, synthesis and evaluation of transition metal containing combinatorial libraries has received much attention in the past few years. As a result, a variety of synthetic techniques have been developed, and rapid assays for metal ion binding have yielded new ligand classes displaying high affinity and selectivity. Research in catalysis has centered around lead optimization using much smaller parallel libraries because of the lack of a truly efficient reaction screening method. Materials science applications have also focused on spatially addressed libraries and have employed a variety of techniques to identify compounds with desired physical properties. Nonetheless, high-throughput characterization and reaction product detection methods must still be developed in order to realize the full potential of combinatorial chemistry for the discovery of novel metal-containing compounds.     

Prejudice toward fat people: the development and validation of the antifat attitudes test

BACKGROUND: The identification of potent small molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful new tools that can be used in these efforts are combinatorial chemistry and structure-based design. Here we address how to join these methods in a design protocol that produces libraries of compounds that are directed against specific macromolecular targets. The aspartyl class of proteases, which is involved in numerous biological processes, was chosen to demonstrate this effective procedure. RESULTS: Using cathepsin D, a prototypical aspartyl protease, a number of low nanomolar inhibitors were rapidly identified. Although cathepsin D is implicated in a number of therapeutically relevant processes, potent nonpeptide inhibitors have not been reported previously. The libraries, synthesized on solid support, displayed nonpeptide functionality about the (hydroxyethyl)amine isostere. The (hydroxyethyl)amine isostere, which targets the aspartyl protease class, is a stable mimetic of the tetrahedral intermediate of amide hydrolysis. Structure-based design, using the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin, was used to select the building blocks for the library synthesis. The library yielded a 'hit rate' of 6-7% at 1 microM inhibitor concentrations, with the most potent compound having a Ki value of 73 nM. More potent, nonpeptide inhibitors (Ki = 9-15 nM) of cathepsin D were rapidly identified by synthesizing and screening a small second generation library. CONCLUSIONS: The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.     

Directed evolution of enzyme catalysts

Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution in vitro includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.     

Covalent immobilization of recombinant human alphavbeta3 integrin on a solid support with retention of functionality

Alphavbeta3 is the major receptor mediating the attachment of osteoclasts to bone surface and plays a critical role in bone resorption and remodeling. Interfering with alphavbeta3 binding inhibits osteoclast-mediated bone resorption, and thus demonstrates the potential utility of alphavbeta3 antagonists for therapy of osteoporosis. This report describes the generation of an alphavbeta3 affinity column which was created to enable screening of collections of large numbers of ligands, e.g., combinatorial libraries (previously prepared by us), to sort and identify ligands with the highest affinity for alphavbeta3. We demonstrate that covalent immobilization of the heterodimeric alphavbeta3 receptor can be achieved with retention of characteristic ligand binding properties. Human alphavbeta3 was isolated from human embryonic kidney cells (HEK 293) that stably express high levels of the recombinant receptor and then affinity purified to homogeneity. Purified alphavbeta3 receptor was linked covalently to activated CH-Sepharose 4B beads. Quantification of immobilized functional receptor was determined by Scatchard analysis. The immobilized functional receptor maintains binding properties similar to the membrane-embedded and soluble receptor. The immobilized receptor also can be used to select the highest affinity ligand from among a mixture of peptides which differ in their binding affinity, structure, and hydrophobicity, both when the peptides are loaded in equimolar concentrations in a mixture and when the concentration of the highest affinity ligand is reduced 10-fold. Liquid chromatography-mass spectrometry was utilized to confirm selective ligand binding and to demonstrate that preferential binding was not due to nonspecific hydrophobic interactions with immobilized alphavbeta3 receptor or the affinity column. This approach may be of general use for affinity-based screening of ligands for other integrins and should enable practical screening of combinatorial libraries containing large numbers of potential ligands for the human alphavbeta3 integrin receptor, including linear peptides, cyclic peptides, and peptidomimetics.     

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-alphav beta3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to alphav beta3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.     

Screening lab tests of Chlamydia trachomatis. Do they show declining trend of the infections?

Peptides are flexible molecules and can adopt local structural features of protein, such as secondary structure, hydrophobicity, and distribution of electrostatic charges, and so forth, and mimic their functions. Therapeutic peptidomimetics that are immunologically relevant are developed by engineering the surface loop structures in the proteins and receptors. The class of molecules targeted include immunoglobulin fold-containing molecules: antibody, cell-surface CD4 receptors and cystine-knot-containing receptor family members: tumor necrosis factor (TNF), CD40, and p185/Neu receptors. We have used the loops involved in the molecular recognition as a template and developed peptidomimetics that interfere with the functions of the target molecules. In this article, two molecular targets are discussed: (1) immunoglobulin fold-containing CD4 receptor and (2) cystine-knot-containing TNF receptor (TNFR).     

Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds

Modified dipeptides have been used successfully for the generation of a variety of small organic and heterocyclic combinatorial libraries, including linear urea, polyamine, hydantoin, thiohydantoin, cyclic urea, cyclic thiourea and bicyclic guanidine. The synthesis and screening results for a number of these libraries are described. The solid phase synthesis of heterocyclic compounds such as diazepine and thiomorpholinone are also described.     

Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins

Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.     

Combinatorial drug discovery: which methods will produce the greatest value?

Combinatorial strategies are important new approaches to drug discovery, and it seems quite likely that they will result in the discovery of interesting potential pharmaceuticals. However, it is less clear whether combinatorial approaches will result in quantum advances in therapeutics. Nor is there general agreement about the factors most important in defining how combinatorial strategies will provide value to the discovery of lead and therapeutic compounds. In this review, we propose criteria that define the value of combinatorial strategies and categorize the various approaches by: (a) the type of chemical space to be searched, (b) the tactics employed to synthesize and screen libraries, and (c) the structures of individual molecules in libraries. We evaluate the strengths and weaknesses of the various strategies and suggest milestones that can help to track their success.     

The search for orally active medications through combinatorial chemistry

The literature of combinatorial chemistry is reviewed with particular attention paid to considerations of absorption, distribution, metabolism and excretion in the design and evaluation of libraries containing drug-like molecules. Published libraries are evaluated in particular for the likelihood that the products would possess oral bioavailability.     

Skin colonization of Staphylococcus aureus in atopic dermatitis patients seen at the National Skin Centre, Singapore

BACKGROUND: Combinatorial chemistry has become an important method for identifying effective ligand-receptor binding, new catalysts and enzyme inhibitors. In order to distinguish the most active component of a library or to obtain structure-activity relationships of compounds in a library, an efficient quantitative assay is crucial. Electrospray mass spectrometry has become an indispensable tool for qualitatively screening combinatorial libraries and its use for quantitative analysis has recently been demonstrated. RESULTS: This paper describes the use of quantitative electrospray mass spectrometry for screening libraries of inhibitors of enzymatic reactions, specifically the enzymatic glycosylation by beta-1,4-galactosyltransferase, which catalyzes the transfer of galactose from uridine-5'-diphosphogalactose to the 4-position of N-acetylglucosamine beta OBn (Bn: benzene) to form N-acetyllactosamine beta OBn. Our mass spectrometric screening approach showed that both nucleoside diphosphates and triphosphates inhibited galactosyltransferase while none of the nucleoside monophosphates, including uridine-5'-monophosphate, showed any inhibition. Additional libraries were generated in which the concentrations of the inhibitors were varied and, using mass spectrometry, uridine-5'-diphosphate-2-deoxy-2-fluorogalactose was identified as the best inhibitor. CONCLUSIONS: This report introduces quantitative electrospray mass spectrometry as a rapid, sensitive and accurate quantitative assaying tool for inhibitor libraries that does not require a chromophore or radiolabeling. A viable alternative to existing analytical techniques is thus provided. The new technique will greatly facilitate the discovery of novel inhibitors against galactosyltransferase, an enzyme for which there are few potent inhibitors.     

Transit Route Network Design Using Parallel Genetic Algorithm

A transit route network design (TRND) problem for urban bus operation involves the determination of a set of transit routes and the associated frequencies that achieve the desired objective. This can be formulated as an optimization problem of minimizing the total system cost, which is the sum of the operating cost and the generalized travel cost. A review of previous approaches to solve this problem reveals the deficiency of conventional optimization techniques and the suitability of genetic algorithm (GA) based models to handle such combinatorial optimization problems. Since GAs are computationally intensive optimization techniques, their application to large and complex problems is limited. The computational performance of a GA model can be improved by exploiting its inherent parallel nature. Accordingly, two parallel genetic algorithm (PGA) models are proposed in this study. The first is a global parallel virtual machine (PVM) parallel GA model where the fitness evaluation is done concurrently in a parallel processing environment using PVM libraries. The second is a global message passing interface (MPI) parallel GA model where an MPI environment substitutes for the PVM libraries. An existing GA model for TRND for a large city is used as a case study. These models are tested for computation time, speedup, and efficiency. From the study, it is observed that the global PVM model performed better than the other model.     

Thyrotropin releasing hormone analogs: a building block approach to the construction of tetracyclic peptidomimetics

A building block based approach was used to synthesize a pair of tetracyclic peptidomimetics that constrain all but one of the rotational degrees of freedom of the hypothalamic tripeptide hormone thyroliberin. One of the analogs bound to the thyroliberin endocrine receptor (TRH-R) with an affinity greater than that of an analog without constraints. The tetracyclic peptidomimetics were found to be partial agonists for the TRH-R receptor.     

Evaluation of glycogen level in human lung carcinoma tissues by an infrared spectroscopic method

Extensive mapping studies for seven antigen-antibody interactions have been carried out using both individual analogs and peptide libraries. With competitive ELISA, these studies have revealed that monoclonal antibodies exhibit a broad range of specificities, from antibodies that recognize only conservative substitutions for 1-2 positions of the antigenic determinant, to antibodies that recognize sequences that are completely unrelated to the parent antigen with comparable affinities. Synthetic combinatorial libraries, containing millions of peptide sequences, permit a more systematic and rapid evaluation of the extent of multiple-binding specificities of monoclonal antibodies than individual analogs. The peptide libraries used here comprise mixtures of compounds having specifically defined positions and mixture positions. The same diversity of sequences in different formats, which differ by the numbers of positions singularly defined and different locations defined within the sequence, can be examined. Comparison of the screening results, selection criteria of the most active mixtures, and different approaches used for the deconvolution of active individual compounds are discussed. Synthetic combinatorial libraries greatly facilitate the understanding of antigen-antibody interactions at the amino acid level and will assist in the development of improved immunodiagnostics.     

Analysis of colorectal cancer by comparative genomic hybridization: evidence for induction of the metastatic phenotype by loss of tumor suppressor genes

A method is described for measuring the diversity of combinatorial oligonucleotide libraries that entails extrapolating the base composition of a co-synthesized model library (dNC, N = A, C, G, T) to that of a multibase library template. The base composition of dNC was measured by HPLC. The ability of dNC to predict the base composition of a multibase library template was corroborated by measuring the composition of a 12 base combinatorial library. The base composition of the 12 base library was determined by several template dependent incorporation assays: measurement of restriction fragment specific activities from polymerase incorporation/restriction enzyme digests, template directed radionucleotide primer extension and quantitative dideoxynucleotide sequencing. Additionally, a convention for describing oligomeric combinatorial library (OCL) diversity is proposed. The convention uses a quantity termed the diversity quotient (Qd) to describe library breadth and the mole fraction of the least represented monomeric unit of the OCL to calculate minimum library quantity requirements. Similar methods/conventions could presumably be developed/adopted for non-nucleic acid libraries.     

Detection of human antibodies using "convergent" combinatorial peptide libraries or "mixotopes" designed from a nonvariable antigen: application to the EBV viral capsid antigen p18

We have previously described the use of synthetic combinatorial "convergent" libraries, or "mixotopes" as immunogens or as antigens to represent naturally hypervariable sequences. The success of this approach suggests that such a mixture of closely related peptides could, at least in part, conveniently represent a nonvariable epitope during its multiple interactions with an antibody population. To address this possibility, we have designed from a non-variable immunodominant peptide of the EBV-viral capsid antigen of 18 kD (VCAp18) a series of three mixotopes containing from 65,000 to 16 million combinatorial sequences. The reactivity of VCAp18 and its three derived mixotopes was examined in ELISA towards a collection of 74 human sera from documented EBV-negative or EBV-positive donors, and analyzed in terms of sensitivity and specificity. Following the observation that the two least degenerated mixotopes could improve the sensitivity of detection of some sera of low reactivity for VCAp18, we decided to combine each mixotope with the VCAp18 peptide. In the case of the least degenerated mixotope in combination with VCAp18, sensitivity and specificity for immunoenzymatic EBV-serodiagnosis, were enhanced to 100%. Our results suggest that synthetic "convergent" combinatorial peptide libraries or "mixotopes," designed from nonvariable antigens, could be useful adjuncts to an antigenic single-sequence peptide in immunoenzymatic serodiagnosis.     

Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.     

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry

A model experiment for the 'on-line' screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and component of a H-gamma-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of 'two-dimensional' screening of substrate libraries by ESI-FTICR mass spectrometry.     

溅射法镀二氧化钛薄膜靶材及工艺研究进展

介绍了溅射法镀膜的基本原理，阐述了溅射法镀二氧化钛薄膜用靶材及相应溅射镀膜工艺的研究现状。溅射镀Tio2薄膜用靶材主要有金属钛、二氧化钛和“非化学计量”二氧化钛靶材3大类。由于钛的氧化态及靶材导电性不同，它们对溅射工艺（如溅射气氛等）有一定具体的要求，通过对靶材和相应工艺进行分析比较，指出应用“非化学计量”靶材是溅射法制备二氧化钛薄膜技术发展的重要方向。