Modern methods to produce natural-product libraries

Natural sources offer a wealth of chemically diverse compounds that have been evolutionary preselected to modulate biochemical pathways. Several industrial and academic groups are accessing this source using advanced technology platforms. Methods have been reported to generate large and diverse natural-product libraries optimised for high-throughput screening and for a fast discovery process. In addition to prefractionated and pure natural-product libraries, parallel synthesis gives access to synthetic, semi-synthetic and natural-product-like libraries. Natural-product chemistry and organic synthesis are powerful tools for optimising natural leads and for generating new diversity from natural scaffolds. The amalgamation of both may be expected to become an important strategy in future drug design.     

Natural products as a screening resource

Natural products have been the most productive source of leads for new drugs, but they are currently out of fashion with the pharmaceutical industry. New approaches to sourcing novel compounds from untapped areas of biodiversity coupled with the technical advances in analytical techniques (such as microcoil NMR and linked LC-MS-NMR) have removed many of the difficulties in using natural products in screening campaigns. As the 'chemical space' occupied by natural products is both more varied and more drug-like than that of combinatorial chemical collections, synthetic and biosynthetic methods are being developed to produce screening libraries of natural product-like compounds. A renaissance of drug discovery inspired by natural products can be predicted.     

Natural products from synthetic biology

DNA sequencing has uncovered microbial secondary metabolic potential that never surfaced in fermentation based screens. Deep and cheap sequencing of a genus such as Streptomyces can rapidly expose hundreds of metabolic genes and operons. Meanwhile, synthetic biologists, in their quest to engineer advanced biofuels, are mastering metabolic engineering. Natural products, a reliable source of new therapeutic leads for many years, have fallen into disfavor with drug discoverers partly because these molecules are rarely available as pure compounds and sourcing is often problematic. The convergence of next generation sequencing and synthetic biology, along with less spectacular progress in analytic technologies such as mass spectroscopy, opens the door to the creation of large, reliable libraries of pure natural products for drug discovery.     

Understanding differences between synthetic and natural antibodies can help improve antibody engineering

Synthetic libraries are a major source of human-like antibody (Ab) drug leads. To assess the similarity between natural Abs and the products of these libraries, we compared large sets of natural and synthetic Abs using “CDRs Analyzer,” a tool we introduce for structural analysis of Ab-antigen (Ag) interactions. Natural Abs, we found, recognize their Ags by combining multiple complementarity-determining regions (CDRs) to create an integrated interface. Synthetic Abs, however, rely dominantly, sometimes even exclusively on CDRH3. The increased contribution of CDRH3 to Ag binding in synthetic Abs comes with a substantial decrease in the involvement of CDRH2 and CDRH1. Furthermore, in natural Abs CDRs specialize in specific types of non-covalent interactions with the Ag. CDRH1 accounts for a significant portion of the cation-pi interactions; CDRH2 is the major source of salt-bridges and CDRH3 accounts for most hydrogen bonds. In synthetic Abs this specialization is lost, and CDRH3 becomes the main sources of all types of contacts. The reliance of synthetic Abs on CDRH3 reduces the complexity of their interaction with the Ag: More Ag residues contact only one CDR and fewer contact 3 CDRs or more. We suggest that the focus of engineering attempts on CDRH3 results in libraries enriched with variants that are not natural-like. This may affect not only Ag binding, but also Ab expression, stability and selectivity. Our findings can help guide library design, creating libraries that can bind more epitopes and Abs that better mimic the natural antigenic interactions.     

New aspects of natural products in drug discovery

During the past 15 years, most large pharmaceutical companies have decreased the screening of natural products for drug discovery in favor of synthetic compound libraries. Main reasons for this include the incompatibility of natural product libraries with high-throughput screening and the marginal improvement in core technologies for natural product screening in the late 1980s and early 1990 s. Recently, the development of new technologies has revolutionized the screening of natural products. Applying these technologies compensates for the inherent limitations of natural products and offers a unique opportunity to re-establish natural products as a major source for drug discovery. Examples of these new advances and technologies are described in this review.     

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace? SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.     

Design of Array-Type Compound Libraries that Combine Information from Natural Products and Synthetic Molecules

A new approach to the design of compound libraries, named MetaFocus (Metabolite-Focused library), is presented that exploits information encoded in natural molecules and combines naturally occurring and synthetic compounds. An important goal of the MF approach is the identification of synthetic compounds that mimic properties of natural molecules that are difficult to obtain in sufficient quantities or to synthesize. Compounds in MetaFocus (MF) arrays are focused on natural molecules with attractive therapeutic effects. Similarity search and diversity design techniques are employed to generate compound arrays that start from a selected natural molecule, add similar molecules, either from natural or synthetic sources, and diversify scaffolds derived from these molecules. Since the identification of similar molecules from natural and synthetic sources plays a significant role in our library design efforts, the performance of fingerprint-type search tools was systematically assessed in a newly assembled test database consisting of 16 biological activity classes. MF arrays are organized as an easily expandable and searchable data structure and serve as a knowledge base for drug discovery applications. Here we introduce the design principles and organization of MF arrays and present example applications.     

Lead compounds discovered from libraries: part 2

Many lead compounds with the potential to progress to viable drug candidates have been identified from libraries during the past two years. There are two key strategies most often employed to find leads from libraries: first, high-throughput biological screening of corporate compound collections; and second, synthesis and screening of project-directed libraries (i.e. target-based libraries). Numerous success stories, including the discovery of several clinical candidates, testify to the utility of chemical library collections as proven sources of new leads for drug development.     

Front‐Loading Natural‐Product‐Screening Libraries for log P: Background,Development, and Implementation

In the period from January 1981 to December 2010, 1068 small‐molecule new chemical entities (NCEs) were introduced, of which ca. 34% are either a natural product or a close analogue. While this metric reflects the impact natural products have played in delivering new chemical starting points (leads) for the pharmaceutical industry, it does not capture the decline this approach has suffered over the last 20 years as the high‐throughput screening (HTS) of pure compound libraries has become more popular. An impediment to natural‐product drug discovery in the HTS paradigm is the lack of a clear strategy that enables front‐loading of an extract or fraction's chemical constituents so that they are compliant with lead‐ and drug‐like chemical space. To address this imbalance, an approach based on lipophilicity, as measured by clog P has been developed that, together with advances being made in isolation and structural elucidation, can afford natural product leads in timelines compatible with pure compound screening.     

A multidimensional overpressured layer chromatographic method for the characterization of tetrazine libraries

The recent combinatorial approach in synthetic organic chemistry started a new age in drug discovery. The generation of compound libraries in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Characterization and separation of such pool of compounds have been lagging behind the synthetic and screening methodologies. Overpressured layer chromatography (OPLC) is an instrumentalized planar liquid chromatographic technique associated with the use of optimized layers prepared from particles of narrow particle size distribution and small diameter. On one hand, uni-directional OPLC allows the simultaneous separation of large number of samples in minutes. On the other hand, two-dimensional OPLC offers multidimensional separation on a single layer. This paper shows the complete multidimensional separation of a tetrazine library prepared by parallel combinatorial synthesis. In general, this approach may become the method of choice for the characterization of compound libraries.     

DNA assembly techniques for next-generation combinatorial biosynthesis of natural products

Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis.     

Unlocking the potential of natural products in drug discovery

The natural product specialized metabolites produced by microbes and plants are the backbone of our current drugs. Despite their historical importance, few pharmaceutical companies currently emphasize their exploitation in new drug discovery and instead favour synthetic compounds as more tractable alternatives. Ironically, we are in a Golden Age of understanding of natural product biosynthesis, biochemistry and engineering. These advances have the potential to usher in a new era of natural product exploration and development taking full advantage of the unique and favourable properties of natural products compounds in drug discovery.     

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions

A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.     

Selectide Technology: Bead-Binding Screening

The Selectide process is a random synthetic chemical library method based on the one-bead one-peptide (structure) concept. A "split-synthesis" method is used to generate huge random libraries (10⁶-10⁸). At the end of the synthesis, each bead expresses only one chemical entity (e.g., peptide). The whole library is then tested simultaneously for binding to a specific acceptor molecule of biologic interest. The ligand bead that interacts specifically with the acceptor molecule is then isolated for structure determination. Once a binding motif is identified, a secondary library (based on the motif of the primary screen) is generated and screened under a more stringent condition to identify leads of higher affinity. This process can be applied to both peptide and nonpeptide (small organic) libraries. In the case of nonsequencable structure libraries, the coding principle has to be applied for structure elucidation of positively reacting beads. Coding peptide is synthesized in parallel to the screening structure, and classical Edman degradation (one or multiple-step) is used for structural analysis. To exclude the possibility of interaction of the macromolecular target (e.g., receptor, enzyme, antibody) with the coding structure, a synthetic technique for segregation of the surface (screening structure) and the interior (coding structure) of the beads was developed. The one-bead one-structure process is invaluable in drug discovery for lead identification as well as further optimization of the initial leads. It also serves as an important research tool for molecular recognition.     

Natural product derivatives with bactericidal activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis

We have shown that the intentional engineering of a natural product biosynthesis pathway is a useful way to generate stereochemically complex scaffolds for use in the generation of combinatorial libraries that capture the structural features of both natural products and synthetic compounds. Analysis of a prototype library based upon nonactic acid lead to the discovery of triazole-containing nonactic acid analogs, a new structural class of antibiotic that exhibits bactericidal activity against drug resistant, Gram-positive pathogens including Staphylococcus aureus and Enterococcus faecalis.     

Systematics-guided bioprospecting for bioactive microbial natural products

Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.     

A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules

Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.     

Synthetic antibodies: Concepts, potential and practical considerations

The last 100years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories.     

Allenic and cumulenic lipids

Nowadays, about 200 natural allenic metabolites, more than 2700 synthetic allenic compounds, and about 1300 cumulenic structures are known. The present review describes research on natural as well as some biological active allenic and cumulenic lipids and related compounds isolated from different sources. Intensive searches for new classes of pharmacologically potent agents produced by living organisms have resulted in the discovery of dozens of such compounds possessing high anticancer, cytotoxic, antibacterial, antiviral, and other activities. Known allenic and cumulenic compounds can be subdivided on several structural classes: fatty acids, hydrocarbons, terpenes, steroids, carotenoids, marine bromoallenes, peptides, aromatic, cumulenic, and miscellaneous compounds. This review emphasizes the role of natural and synthetic allenic and cumulenic lipids and other related compounds as an important source of leads for drug discovery.     

Will natural products remain an important source of drug research for the future?

In the highly competitive environment of contemporary pharmaceutical research, natural products provide a unique element of molecular diversity and biological functionality which is indispensable for drug discovery. The emergence of strategies to deliver drug leads from natural products within the same time frame as synthetic chemical screening has eliminated a major limitation of the past. At a more functional level, the application of molecular genetics techniques has permitted the manipulation of biosynthetic pathways for the generation of novel chemical species as well as rendering hitherto uncultivatable microorganisms accessible for secondary metabolite generation. These developments augur well for an industry confronted with the challenge of finding lead compounds directed at the plethora of new targets arising from genomics projects. The exploitation of structural chemical databases comprising a wide variety of chemotypes, in conjunction with databases on target genes and proteins, will facilitate the creation of new chemical entities through computational molecular modelling for pharmacological evaluation.