DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.     

Combinatorial search for advanced luminescence materials.

Phosphors are key materials in fluorescent lighting, displays, x-ray scintillation, etc. The rapid development of modern photonic technologies, e.g., mercury-free lamps, flat panel displays, CT-detector array, etc., demands timely discovery of advanced phosphors. To this end, a combinatorial approach has been developed and applied to accelerated experimental search of advanced phosphors and scintillators. Phosphor libraries can be made in both thin film and powder form, using masking strategies and liquid dispensing systems, respectively. High-density libraries with 100 to 1000 discrete phosphor compositions on a 1"-square substrate can be made routinely. Both compositions and synthesis temperatures can be screened in a high-throughput mode. In this article, details on the existing methods of combinatorial synthesis and screening of phosphors will be reported with examples. These methods are generic tools for application of combinatorial chemistry in the discovery of other solid state materials. A few highly efficient phosphors discovered with combinatorial methods have been reproduced in bulk form and their luminescent properties measured.     

Design, docking, and evaluation of multiple libraries against multiple targets

We present a general approach to the design, docking, and virtual screening of multiple combinatorial libraries against a family of proteins. The method consists of three main stages: docking the scaffold, selecting the best substituents at each site of diversity, and comparing the resultant molecules within and between the libraries. The core "divide-and-conquer" algorithm for side-chain selection, developed from an earlier version (Sun et al., J Comp Aided Mol Design 1998;12:597-604), provides a way to explore large lists of substituents with linear rather than combinatorial time dependence. We have applied our method to three combinatorial libraries and three serine proteases: trypsin, chymotrypsin, and elastase. We show that the scaffold docking procedure, in conjunction with a novel vector-based orientation filter, reproduces crystallographic binding modes. In addition, the free-energy-based scoring procedure (Zou et al., J Am Chem Soc 1999;121:8033-8043) is able to reproduce experimental binding data for P1 mutants of macromolecular protease inhibitors. Finally, we show that our method discriminates between a peptide library and virtual libraries built on benzodiazepine and tetrahydroisoquinolinone scaffolds. Implications of the docking results for library design are explored.     

Optimization of chemical libraries by neural networks

Neural networks are finding ever-more applications in the design of combinatorial libraries. These can be divided into two types: Kohonen (self-organizing) maps, and feed-forward networks. While the number of applications is currently quite limited, a rapid increase in publications in this area can be expected in the next few years from the rapid development of general combinatorial chemistry technology.     

Symmetric building blocks and combinatorial functional group transformation as versatile strategies in combinatorial chemistry

A combination of symmetric building blocks and combinatorial functional group transformation for synthesis of pyrimidines was investigated. The purpose of the study was to maximize the return on invested synthetic efforts of reaction development for libraries. A representative set of symmetric diacids was coupled onto deprotected TentaGel Rink Amide resin. The amino function served as a model of a chemical process providing a functional group for additional synthetic steps, while the symmetric building blocks served as a model to connect synthesis protocols and to switch to a different synthesis paradigm consecutively. The reaction sequence was continued in a noncombinatorial step by coupling a bifunctional reagent (3-aminoacetophenone) to the remaining carboxy function of the symmetric diacid. The ketone served as a model of a reagent prepared for combinatorial functional group transformation. The arylmethylketone was reacted with a set of aryl- and heteroarylaldehydes to give alpha,beta-unsaturated ketones. Subsequently, guanidine, alkyl-, and arylcarboxamidines were introduced in combinatorial synthesis of substituted pyrimidines by reaction with the alpha, beta-unsaturated ketone functionality. The combination of symmetric building blocks and combinatorial functional group transformation created a versatile reaction sequence ideally suited for production of libraries from libraries with added diversity.     

Host-guest chemistry: combinatorial receptors.

A combinatorial approach to receptor design provides an expedient method to discover the most effective host-guest complexes from within a library. Recent advances focus on generation of larger libraries, facile detection, combinatorial catalysis and the formation of dynamic receptor libraries.     

Structure‐based design of combinatorial mutagenesis libraries

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high‐throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general‐purpose method, called “Structure‐based Optimization of Combinatorial Mutagenesis ” (SOCoM ), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library‐averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β‐lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure‐based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large‐scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure‐based assessments, such as the energy gap between alternative conformational or bound states.     

Discovery of high-affinity peptide ligands for vancomycin

Vancomycin, an important antibiotic against medically relevant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, exerts its antibacterial effects by binding with moderate affinity to the C-terminal Lys-D-Ala-D-Ala motif (Kaa) of the bacterial cell wall peptide precursor. Essential for Kaa binding to vancomcyin is the free-carboxyl group on the terminal D-Ala in Kaa. In efforts to identify other Kaa-based peptides which bind vancomycin with higher affinity, we utilized our one-bead-one-compound (OBOC) combinatorial library approach, a method which has been widely used to discover highly specific ligands against various receptors. In standard OBOC peptide libraries, the C-terminal end of the synthesized peptide is tethered to a solid-support/resin, however, this study reports development of a synthetic strategy for generating OBOC peptide libraries with a free D-Ala-D-Ala carboxyl end. We screened these "OBOC inverted" peptide libraries against vancomycin, and discovered a series of peptide ligands with strong consensus, which bind vancomycin. To further optimize these ligands, two highly focused Kaa-containing OBOC combinatorial peptidomimetic libraries were designed, synthesized, and screened against vancomycin under more stringent conditions. Peptidomimetic ligands which bind vancomycin with higher affinity than Kaa were identified. The dissociation constant of one of these ligands, Lys(Ac)-HOCit-Glu-Cha-Lys(3,5-dihydroxybenzoyl)-D-Ala-D-Ala (9), as determined by surface plasmon resonance, was 1.03 microM, roughly a 50-fold improvement in affinity compared to Kaa (K(D) = 50 microM).     

T-cell epitope prediction with combinatorial peptide libraries.

T cell receptors (TCR) recognize antigenic peptides in complex with the major histocompatibility complex (MHC) molecules and this trimolecular interaction initiates antigen-specific signaling pathways in the responding T lymphocytes. For the study of autoimmune diseases and vaccine development, it is important to identify peptides (epitopes) that can stimulate a given TCR. The use of combinatorial peptide libraries has recently been introduced as a powerful tool for this purpose. A combinatorial library of n-mer peptides is a set of complex mixtures each characterized by one position fixed to be a specified amino acid and all other positions randomized. A given TCR can be fingerprinted by screening a variety of combinatorial libraries using a proliferation assay. Here, we present statistical models for elucidating the recognition profile of a TCR using combinatorial library proliferation assay data and known MHC binding data.     

Combinatorial synthesis of natural products

Combinatorial syntheses allow production of compound libraries in an expeditious and organized manner immediately applicable for high-throughput screening. Natural products possess a pedigree to justify quality and appreciation in drug discovery and development. Currently, we are seeing a rapid increase in application of natural products in combinatorial chemistry and vice versa. The therapeutic areas of infectious disease and oncology still dominate but many new areas are emerging. Several complex natural products have now been synthesised by solid-phase methods and have created the foundation for preparation of combinatorial libraries. In other examples, natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules. Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which facilitates the creation of combinatorial libraries.     

The dragon on the gold: myths and realities for data mining in biomedicine and biotechnology using digital and molecular libraries

To develop bioscience and personalized medicine in the post-genomic era, the biggest problem may be how to extract knowledge from the rich libraries of biomedical data. A particular dragon protects the gold therein: the dragon is the "curse of dimensionality" and its formidable fire weapon, which is burning researchers, is the "combinatorial explosion". This arises because many genomic, proteomic, clinical, and lifestyle factors may interact that cannot necessarily be considered on a simple pairwise or additive basis. A suggested theoretical solution--or at least "road map" that ameliorates management of these problems--borrows from several disciplines. It is undertaken also in the hope might also lead to research with broader impact on several unresolved issues in biotechnology: conversely, mathematical understanding of processes involving molecular libraries, such as cDNA libraries and DNA in the living cell itself, may open the opportunities to use biotechnology to construct nanotechnological storage and query systems.     

Combinatorial biocatalysis

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.     

Identification of affinity ligands for protein purification from synthetic chemical combinatorial libraries

A method to screen combinatorial libraries for the development of selective ligands for protein affinity chromatographic purification is described. The method is based on the application of parallel combinatorial libraries, and it has several potential advantages. The screening procedure is simple and straightforward, and it does not require the chemical derivatization of the target proteins or even that the target protein be pure. The experiment can also be designed to select binders that are less likely to cause protein denaturation. Feasibility of this approach is demonstrated with a model study of the chromatographic purification of bovine albumin serum (BSA) and Avidin.     

Structure-based and combinatorial search for new RNA-binding drugs

The discovery by structure-based and combinatorial methods of new RNA-binding drugs presents great opportunities for pharmacological development against drug-resistant bacterial and viral pathogens. A handful of recent RNA structures and more numerous studies of the interaction of combinatorial libraries and oligomeric RNA-binding compounds are providing the foundation for effective RNA-targeted drug discovery programs.     

A road less traveled by: exploring a decade of Ellman chemistry

The Ellman group has been one of the most influential in the development and widespread adoption of combinatorial chemistry techniques for biomedical research. Their work has included substantial methodological development for library synthesis with a particular focus on new scaffolds rationally targeted to biomolecules of interest and biologically relevant natural products. Herein we analyze a representative set of libraries from this group with respect to their biological and biomedical relevance in comparison to existing drugs and probe compounds. This analysis reveals that the Ellman group has not only provided new methodologies to the community but also provided libraries with unique potential for further biological study.     

Combinatorial biocatalysis: taking the lead from nature

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.     

Tools for genome-wide strain design and construction

Advances in DNA sequencing and synthesis technologies concurrent with the development of new recombinant DNA approaches have enabled the extension of directed evolution algorithms to the genome-scale. It is now possible to simultaneously map the effect of mutation(s) in each and every gene in the genome onto almost any screenable or selectable phenotype in less than a week. Such maps can be used to direct the design and construction of libraries containing billions of rationally designed combinatorial mutations. Such combinatorial libraries can now also be created and evaluated in less than a week. The review presents and discusses these new technologies within the context of directed evolution and inverse metabolic engineering.