DNA fragmentation based combinatorial approaches to soluble protein expression Part II: library expression, screening and scale-up

In this second of a two-part review encompassing random, combinatorial methods for soluble protein 'domain hunting', we focus upon the expression screening from DNA fragment libraries. Given a library of domain length-encoding DNA fragments assembled in expression vectors, it is necessary to devise reliable means to screen the sample DNA fragment population to find those that express stable, soluble target protein fragments, suitable for the required downstream aims. This review summarizes a variety of alternative strategies that have been employed to identify such stable truncates of full-length proteins. In addition, we review measures that can determine the quality of the expressed protein, the likely reliability of these measures, and the apparent extent of their application within the featured studies.     

Inhibitors of bacterial two-component signalling systems

Bacterial two-component regulatory systems (TCS) play a pivotal role in the process of infection. These signal transduction systems enable bacterial pathogens to mount an adaptive response and cope with diverse environmental stresses, including nutrient deprivation, antibiotic onslaught and phagocytosis. Interest in these systems as novel bacterial targets has been rekindled by the recent discovery of several essential systems in important Gram-positive and Gram-negative pathogens. Several series of TCS inhibitors derived from broad screening approaches have been reported in the literature, however, most appear to suffer from poor selectivity, excessive protein binding and/or limited bioavailability. Consequently, pharmaceutical chemists have turned to alternate strategies, such as the design of substrate-based inhibitors, the generation of combinatorial libraries and the isolation of natural products, to identify inhibitors with more desirable properties. Recent structural studies of the histidine protein kinase and response regulator proteins that constitute TCS may provide a foundation for a structure-based design approach to TCS inhibitors.     

Inhibitors of bacterial two-component signalling systems

Bacterial two-component regulatory systems (TCS) play a pivotal role in the process of infection. These signal transduction systems enable bacterial pathogens to mount an adaptive response and cope with diverse environmental stresses, including nutrient deprivation, antibiotic onslaught and phagocytosis. Interest in these systems as novel bacterial targets has been rekindled by the recent discovery of several essential systems in important Gram-positive and Gram-negative pathogens. Several series of TCS inhibitors derived from broad screening approaches have been reported in the literature, however, most appear to suffer from poor selectivity, excessive protein binding and/or limited bioavailability. Consequently, pharmaceutical chemists have turned to alternate strategies, such as the design of substrate-based inhibitors, the generation of combinatorial libraries and the isolation of natural products, to identify inhibitors with more desirable properties. Recent structural studies of the histidine protein kinase and response regulator proteins that constitute TCS may provide a foundation for a structure-based design approach to TCS inhibitors.     

Peptide aptamers as guides for small-molecule drug discovery

Peptide aptamers are combinatorial protein reagents that bind to target proteins with a high specificity and a strong affinity. By so doing, they can modulate the function of their cognate targets. Because peptide aptamers introduce perturbations that are similar to those caused by therapeutic molecules, their use identifies and/or validates therapeutic targets with a higher confidence level than is typically provided by methods that act upon protein expression levels. The unbiased combinatorial nature of peptide aptamers enables them to 'decorate' numerous polymorphic protein surfaces, whose biological relevance can be inferred through characterization of the peptide aptamers. Bioactive aptamers that bind druggable surfaces can be used in displacement screening assays to identify small-molecule hits to the surfaces. The peptide aptamer technology has a positive impact on drug discovery by addressing major causes of failure and by offering a seamless, cost-effective process from target validation to hit identification.     

Combinatorial approaches in anticancer drug discovery: recent advances in design and synthesis

Combinatorial technology for the generation of molecular diversity has evolved as an integrated component in accelerated drug discovery process. During the emerging days of combinatorial chemistry, solid-phase organic synthesis has been the leading strategy for the production of large libraries for lead discovery. As combinatorial techniques for the library synthesis has evolved, solution-phase synthesis of smaller, targeted libraries is gaining attention. Numerous syntheses of biologically active chemical libraries of small molecules have been reported during the past decade. This review will focus only on the recent literature of chemical libraries targeted towards anticancer properties. The synthesis, chemistry and biological activity of these libraries as anticancer agents are summarized.     

Antineoplastic agents from natural sources: achievements and future directions

The influence of natural products upon anticancer drug discovery and design cannot be overestimated. Approximately 60% of all drugs now in clinical trials for the multiplicity of cancers are either natural products, compounds derived from natural products, contain pharmacophores derived from active natural products or are 'old drugs in new clothes', where (modified) natural products are attached to targeting systems. This review covers those materials that the authors are aware of as being in clinical trials through early 2000 and demonstrates how, even today, in the presence of massive numbers of agents from combinatorial libraries, the compounds produced by 'Mother Nature' are still in the forefront of cancer chemotherapeutics as sources of active chemotypes.     

Discovery of antibacterials and other bioactive compounds from microorganisms-evaluating methodologies for discovery and generation of non-ribosomal peptide antibiotics

After decades of neglect in industrial research the comeback of natural products is due since improved screening approaches are at disposal, yielding a multitude of new compounds from natural sources. Besides traditional compound libraries peptides are characterized by an enormous structural complexity, thus increasing the chance of finding a hit in a screening. Emphasizing antibacterial compounds structural complexity is a prerequisite for their success. This review focuses on the screening approaches employed for the discovery of mostly antibacterial, non-ribosomal peptides derived from natural sources. Traditional screening methodologies as well as genetic approaches are discussed in this context. Utilizing genetic engineering methods e.g., precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis, as well as chemoenzymatics to achieve greater structural diversity is thoroughly discussed and exemplified by recent discoveries.     

“Splicing up” drug discovery. : Cell-based expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides

The present paper reviews the use of protein splicing for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes thus providing a new way for finding therapeutic agents.     

Incorporation of heterocycles into the backbone of peptoids to generate diverse peptoid-inspired one bead one compound libraries

Combinatorial libraries of peptoids (oligo-N-substituted glycines) have proven to be useful sources of protein ligands. Each unit of the peptoid oligomer is derived from 2-haloacetic acid and a primary amine. To increase the chemical diversity available in peptoid libraries, we demonstrate here that heterocyclic halomethyl carboxylic acids can be employed as backbone building blocks in the synthesis of peptoid-based oligomers. Optimized conditions are reported that allow the creation of large, high quality combinatorial libraries containing these units.     

Drug discovery by dynamic combinatorial libraries

Dynamic combinatorial chemistry is a recently introduced supramolecular approach that uses self-assembly processes to generate libraries of chemical compounds. In contrast to the stepwise methodology of classical combinatorial techniques, dynamic combinatorial chemistry allows for the generation of libraries based on the continuous interconversion between the library constituents. Spontaneous assembly of the building blocks through reversible chemical reactions virtually encompasses all possible combinations, and allows the establishment of adaptive processes owing to the dynamic interchange of the library constituents. Addition of the target ligand or receptor creates a driving force that favours the formation of the best-binding constituent--a self-screening process that is capable, in principle, of accelerating the identification of lead compounds for drug discovery.     

Antineoplastic agents from natural sources: achievements and future directions

The influence of natural products upon anticancer drug discovery and design cannot be overestimated. Approximately 60% of all drugs now in clinical trials for the multiplicity of cancers are either natural products, compounds derived from natural products, contain pharmacophores derived from active natural products or are ‘old drugs in new clothes’, where (modified) natural products are attached to targeting systems. This review covers those materials that the authors are aware of as being in clinical trials through early 2000 and demonstrates how, even today, in the presence of massive numbers of agents from combinatorial libraries, the compounds produced by ‘Mother Nature’ are still in the forefront of cancer chemotherapeutics as sources of active chemotypes.     

Bacterial DNA replication enzymes as targets for antibacterial drug discovery

Introduction: The bacterial replisome is composed of a large number of enzymes, which work in exquisite coordination to accomplish chromosomal replication. Effective inhibition inside the bacterial cell of any of the ‘essential’ enzymes of the DNA replication pathway should be detrimental to cell survival.

Areas covered: This review covers DNA replication enzymes that have been shown to have a potential for delivering antibacterial compounds or drug candidates including: type II topoisomerases, a clinically validated target family, and DNA ligase, which has yielded inhibitors with in vivo efficacy. A few of the ‘replisome’ enzymes that are structurally and functionally well characterized and have been subjects of antibacterial discovery efforts are also discussed.

Expert opinion: Identification of several essential genes in the bacterial replication pathway raised hopes that targeting these gene products would lead to novel antibacterials. However, none of these novel, single gene targets have delivered antibacterial drug candidates into clinical trials. This lack of productivity may be due to the target properties and inhibitor identification approaches employed. For DNA primase, DNA helicase and other replisome targets, with the exception of DNA ligase, the exploitation of structure for lead generation has not been tested to the same extent that it has for DNA gyrase. Utilization of structural information should be considered to augment HTS efforts and initiate fragment-based lead generation. The complex protein–protein interactions involved in regulation of replication may explain why biochemical approaches have been less productive for some replisome targets than more independently functioning targets such as DNA ligase or DNA gyrase.     

Genetic diversity: applications of evolutionary algorithms to combinatorial library design

Combinatorial chemistry and other high-throughput techniques continue to affect the drug discovery process in a profound manner. This review examines the impact of combinatorial chemistry upon the discipline of computer-aided molecular design and describes the methods that are being developed to enable researchers to quantify molecular diversity and to design efficient combinatorial libraries. In particular, the application of optimisation algorithms based on evolutionary principles to these problems is examined and illustrated with examples from the literature.     

Diversity oriented synthesis and branching reaction pathway to generate natural product-like compounds

Combinatorial chemistry can be used to synthesize diversified molecules on a large scale. As with all large-scale experiments, this process requires a major investment in equipment, consumables and time. Therefore, careful design is critical. As the complexity of the libraries to be generated increases, additional considerations become important. What are the issues that should be considered when planning combinatorial chemistry projects? Which features in the design strategy are critical to consider ensuring that all of the potential products will be synthesized? How are the reactants selected to optimize product synthesis and yield? Over the last several years, through an experimental process, we have successfully developed and optimized our synthetic strategy. Our approach incorporates a number of critical components into a tightly controlled process that generates molecules with maximal structural complexity. This complexity emanates from carbon-carbon bond formation, which is extremely stable and it is reminiscent of complex natural product molecules. Our studies have illustrated that transition metal catalysts are powerful reagents that can be used to drive the synthesis of diverse small molecules from less complex starting materials. In this review, we will describe some of our recent efforts to synthesize natural product-like molecules and their derivative structures to successfully create libraries of complex molecules for drug discovery applications. Our diversity-oriented synthesis methods incorporate transition metal catalysts, as a versatile tool for creating carbon-carbon bonds and structural complexity, and the branched reaction pathway, as a method for incorporating diversity into the molecular scaffolds. We will review our combinatorial chemistry program, focusing on the decisions that we made for (1) the scaffold selection; (2) the design of a diversity oriented approach for library synthesis; (3) the incorporation of the branched reaction pathway to generate natural product-like molecules from the same starting material; and (4) the process steps that we selected for chemistry development and library generation.     

Process technologies for purity enhancement of large discovery libraries

The design, synthesis and characterization of structurally diverse compound libraries are key first steps in drug discovery. High-throughput screening of these libraries against protein targets is a useful procedure for identifying hits, which can then be optimized to produce compound leads against these protein targets. For this process to be most successful the initial compounds screened must be of high quality, necessitating high-throughput characterization and purification. This review summarizes recent advances in purification processes for combinatorial chemistry.     

Combinatorial synthesis of cholesterol ester transfer protein-mRNA ligands and screening by nondenaturating gel-electrophoresis

RNA, as one of the biomolecules with the most structural and functional diversity, is an attractive therapeutic target.(1) Employing combinatorial chemistry methods, small peptide ligands were found, which bind to a short RNA with important biological functions. A 23-nt RNA oligonucleotide from the cholesterol ester transfer protein mRNA was chosen as a molecular target.(2) A 27-nt RNA oligonucleotide from the human immunodeficiency virus type-1 (HIV-1) TAR RNA was used to control the binding specificity.(3) Tetrapeptide libraries, composed of the amino acids Lys, Tyr, Leu, Ile, and Arg, with and without C- and N-terminal lysines, were synthesized by a combination of combinatorial and divergent solid-phase synthesis. Gel-shift affinity screening was used to extract the peptides with the best RNA binding properties. The peptide Lys-Tyr-Lys-Leu-Tyr-Lys-Cys-NH(2) (1) showing micromolar affinity to its RNA target was characterized with circular dichroism (CD), ultra violet (UV) measurement, and (1)H NMR spectroscopy.     

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis is playing an increasingly important role in natural product-based drug discovery and development programmes. This review highlights the requirements and challenges associated with this conceptually simple strategy of using surrogate hosts for the production of natural products in good yields and for the generation of novel analogues by combinatorial biosynthesis methods, taking advantage of the recombinant DNA technologies and tools available in the model hosts. Specific topics addressed include: i) the mobilisation of biosynthetic gene clusters using different vector systems; ii) the selection of suitable model heterologous hosts; iii) the requirement of post-translational protein modifications and precursor supply within the model hosts; iv) the influence of promoters and pathway regulators; and v) the choice of suitable fermentation conditions. Lastly, the use of heterologous expression in combinatorial biosynthesis is addressed. Future directions for model heterologous host engineering and the optimisation of natural product biosynthetic gene cluster expression in heterologous hosts are also discussed.