Beyond Rf tagging

The split-pool diversity orientated synthesis method, which requires some form of encoding to track the synthesis of discrete compounds, has been the lynchpin of most combinatorial synthesis efforts. The use of encoding methods in combinatorial chemistry has matured, and depending on their level of resources, chemists now have a diverse choice of encoding methods available. New methods of encoding have been developed that are inexpensive, simple to incorporate into any laboratory, and utilize analytical equipment such as MS, FTIR and NMR that are readily available to most organic chemists.     

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.     

Diversity assessment.

Several themes have been highlighted recently in both conferences and publications: the availability of product-focused and pharmacophore-based methods for the analysis and design of combinatorial libraries; the power of cell-based methods for molecular similarity, diversity and library design applications; methods for 'rational' diverse subset selection (with applicability to library design); the need for specialized optimization programs for the design of combinatorial libraries that maximize the use of common reagents; and the concept of 'drug-likeness' and its importance in the design of combinatorial libraries.     

Novel miniaturized systems in high-throughput screening

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100,000 compounds per second.     

Branch-and-terminate: a combinatorial optimization algorithm for protein design.

BACKGROUND: Several deterministic and stochastic combinatorial optimization algorithms have been applied to computational protein design and homology modeling. As structural targets increase in size, however, it has become necessary to find more powerful methods to address the increased combinatorial complexity. RESULTS: We present a new deterministic combinatorial search algorithm called 'Branch-and-Terminate' (B&T), which is derived from the Branch-and-Bound search method. The B&T approach is based on the construction of an efficient but very restrictive bounding expression, which is used for the search of a combinatorial tree representing the protein system. The bounding expression is used both to determine the optimal organization of the tree and to perform a highly effective pruning procedure named 'termination'. For some calculations, the B&T method rivals the current deterministic standard, dead-end elimination (DEE), sometimes finding the solution up to 21 times faster. A more significant feature of the B&T algorithm is that it can provide an efficient way to complete the optimization of problems that have been partially reduced by a DEE algorithm. CONCLUSIONS: The B&T algorithm is an effective optimization algorithm when used alone. Moreover, it can increase the problem size limit of amino acid sidechain placement calculations, such as protein design, by completing DEE optimizations that reach a point at which the DEE criteria become inefficient. Together the two algorithms make it possible to find solutions to problems that are intractable by either algorithm alone.     

Microbial pathway engineering for industrial processes: evolution, combinatorial biosynthesis and rational design

Microbial pathway engineering has made significant progress in multiple areas. Many examples of successful pathway engineering for specialty and fine chemicals have been reported in the past two years. Novel carotenoids and polyketides have been synthesized using molecular evolution and combinatorial strategies. In addition, rational design approaches based on metabolic control have been reported to increase metabolic flux to specific products. Experimental and computational tools have been developed to aid in design, reconstruction and analysis of non-native pathways. It is expected that a hybrid of evolutionary, combinatorial and rational design approaches will yield significant advances in the near future.     

Role of dynamic interactions in effective signal transfer for Gbeta stimulation of phospholipase C-beta 2

Heterotrimeric G protein subunits regulate their effectors by protein-protein interactions. The regions involved in these direct interactions have either signal transfer or general binding functions (Buck, E., Li, J., Chen, Y., Weng, G., Scarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). Although key determinants of signal transfer regions for G protein subunits have been identified, the mechanisms of signal transfer are not fully understood. We have used a combinatorial peptide approach to analyze one Gbeta region, Gbeta86-105, involved in signal transfer to the effector phospholipase C (PLC)-beta2 to gain a more mechanistic understanding of Gbeta/PLC-beta2 signaling. Binding and functional studies with the combinatorial peptides on interaction with and stimulation/inhibition of phospholipase Cbeta2 indicate that binding affinity can be resolved from EC(50) for functional effects, such that peptides that have wild type binding affinities have 15- to 20-fold lower EC(50) values. Although more potent, these peptides display a much lower extent of maximal stimulation. These peptides synergize with Gbetagamma or peptides encoding the second Gbeta42-54 signal transfer region in maximally stimulating phospholipase C-beta2. Other combinatorial peptides from the Gbeta86-105 region that bind to PLC-beta2 by themselves submaximally stimulate and extensively inhibit Gbetagamma stimulation of PLC-beta2. The intrinsic stimulation function can be attributed to Arg-96 and Ser-97, the synergy function to Trp-99, and the binding affinity to Thr-87, Val-90, Pro-94, Arg-96, Ser-97, and Val-100. These results indicate that, even within signal transfer regions, residues involved in binding can be resolved from those involved in signal transfer and that signal transfer is likely to be achieved through dynamic rather than steady-state interactions.     

Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library

A combinatorial random protein library was constructed from random DNA fragments generated by "DNA random priming", an improved method of "random-priming recombination" using random-sequence primers and template cDNA from the yeast Saccharomyces cerevisiae. In order to express this library on the yeast cell surface, a yeast multicopy cassette vector was constructed, in which the random-protein-encoding DNA fragments were fused to a gene encoding the C-terminal 320 amino acids of alpha-agglutinin. Fluorescent labeling of the immuno-reaction of RGS(His)(6) epitope confirmed the surface display of random proteins. The surface display of heterologous random proteins on yeast cells will have a wide application. As an example, an n-nonane-tolerant yeast strain that could grow very well in nonane-overlaid culture medium was screened out from transformants displaying this combinatorial library. n-Nonane tolerance was dependent on the transformed plasmid, and the related protein was confirmed to localize on the cell surface by papain treatment and immunofluorescent labeling. Analysis of this displayed protein was also carried out. This strain is the first one to have been endowed artificially with organic solvent tolerance. This is a good example of creating cells exhibiting new phenotypes using a combinatorial protein library.     

Mapping protein-protein interactions with combinatorial biology methods

Extremely diverse, DNA-encoded libraries of peptides and proteins have been constructed that include a linkage between each polypeptide and the encoding DNA. Library members can be selected by virtue of a particular binding specificity, and their protein sequence can be deduced from the sequence of the cognate DNA. Such combinatorial biology methods have proven invaluable in both identifying natural protein-protein interactions and also in mapping the specificities and energetics of these interactions in fine detail.     

Core-directed protein design

For various reasons, it seems sensible to redesign or design proteins from the inside out. Past approaches in this field have involved iterations of mutagenesis and characterisation to 'evolve' designs. Increasingly, combinatorial approaches are being taken to select 'fit' sequences from libraries of variant proteins. In particular, in silico methods have been used to good effect. More recently, experimental methods have been developed and improved. We are now in a position to redesign stability and function into natural protein frameworks confidently and to attempt de novo designs for more ambitious targets.     

Novel methods for directed evolution of enzymes: quality, not quantity

In the past decade methods of directed molecular evolution have proven revolutionary in protein engineering. An increasing number of powerful new combinatorial techniques have joined rational design methods as effective tools for the manipulation and tailoring of biocatalysts. More and more, research in this maturing field is focusing on the quality and comprehensiveness of library construction and analysis. Additionally, in-depth studies have begun to highlight the underlying evolutionary mechanisms, limitations, and consequences of the various methodologies. Together, these investigations are creating a framework for future engineering projects.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

Engineering and applications of genetic circuits

In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.     

'One bead two compound libraries' for detecting chemical and biochemical conversions

When combinatorial chemistry was introduced 13 years ago, the expectations were high for the delivery of results, particularly in the pharmaceutical industry. However, combinatorial chemistry was implemented independently of the application for which the products were going to be used. Resins developed only for efficient solid-phase synthesis were used and products were employed in existing assays developed for traditional solution studies. There was almost no assay or technology development and the use of real combinatorial methods soon had to give way to high-throughput synthesis and traditional screening. However, during recent years more sophisticated resins and assay techniques have been developed that may result in a second and more successful implementation of real integrated combinatorial chemistry. The first in this line of new developments is the 'one bead two compound' assay, in which the resin bead in addition to a combinatorial library member contains a reporter compound that can act as a beacon to monitor the activity of the library member. This powerful concept can be generally applied in all fields of combinatorial chemistry including drug, catalysts and material development.     

Exploring the possibilities presented by protein engineering

A combination of classical and powerful new combinatorial genetic techniques allows the redesign of enzyme activities and creation of proteins that are tailored to have specific properties. These technologies have far-reaching consequences for the future design of crop plants and the storage compounds within them.     

A “Fuzzy”-Logic Language for Encoding Multiple Physical Traits in Biomolecules

To carry out their activities, biological macromolecules balance different physical traits, such as stability, interaction affinity, and selectivity. How such often opposing traits are encoded in a macromolecular system is critical to our understanding of evolutionary processes and ability to design new molecules with desired functions. We present a framework for constraining design simulations to balance different physical characteristics. Each trait is represented by the equilibrium fractional occupancy of the desired state relative to its alternatives, ranging from none to full occupancy, and the different traits are combined using Boolean operators to effect a “fuzzy”-logic language for encoding any combination of traits. In another paper, we presented a new combinatorial backbone design algorithm AbDesign where the fuzzy-logic framework was used to optimize protein backbones and sequences for both stability and binding affinity in antibody-design simulation. We now extend this framework and find that fuzzy-logic design simulations reproduce sequence and structure design principles seen in nature to underlie exquisite specificity on the one hand and multispecificity on the other hand. The fuzzy-logic language is broadly applicable and could help define the space of tolerated and beneficial mutations in natural biomolecular systems and design artificial molecules that encode complex characteristics.     

Murine Cell Glycolipids Customization by Modular Expression of Glycosyltransferases

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.     

Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals

Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed.     

Reagent-based and product-based computational approaches in library design

The design of combinatorial libraries involves the consideration of all synthesizable compounds (the virtual library), followed by the selection of a suitably sized subset for actual synthesis and experimentation. Several approaches to this task can be envisaged, involving either reagent-based or product-based considerations. Reagent-based design considers the properties of the building blocks rather than those of the final products. Although popular with chemists, this approach overlooks the extent of chemical transformations involved in generating products. In effect, several important properties cannot be derived from building blocks alone and require access to product structures. Several studies have demonstrated the superiority of product-based designs in yielding diverse and representative subsets. Although more computationally intensive, the latter approach provides a basis for more sophisticated designs where reagent-based and product based considerations can be combined for a best-of-breed approach.