Current Topics in Medicinal Chemistry - Volume 8, Issue 8, 2008

Natural products synthesized by a variety of micro- and macro-organisms are chemically very diverse, and represent a rich source for drug discovery. These compounds are often not required for growth and maintenance of the organisms producing them, but rather act as chemical weapons or signalling molecules, thereby providing a certain advantage. Whatever is the true biological function of a certain natural product, humans have tried for years to adapt these compounds as medicines to cure various diseases. However, we must be aware of the fact that these compounds have been evolved to serve the organism's own needs, and not to help us in our fight against diseases. It follows then, that in most cases, although displaying desired biological activity, a natural product might not possess drug-like properties required for their successful application in medicine. Such properties as solubility, absorption in the gastrointestinal tract, metabolic and aqueous stability, geno- and cardiotoxicity, pharmacokinetics and pharmacodynamics are all important for consideration of a drug candidate. Some of these properties may be improved by medicinal chemistry through synthesis of natural product analogues. This approach is rather fast and efficient, and can rapidly provide important data on structure-activity relationship. However, many natural products are very complex, and specific chemical modifications are difficult or impossible to achieve. The latter problem may be circumvented by the use of biosynthetic engineering, a relatively new trend that has been developing over the last 20 years in several laboratories. The pre-requisite for biosynthetic engineering of a natural product is molecular cloning of the entire gene set encoding the enzymes involved in its biosynthesis. Current knowledge on basic principles of natural product biosynthesis, along with access to powerful bioinformatics tools and databases allow to deduce complete biosynthetic pathways for many natural products. Once the biosynthetic route for a natural product is established, and genetic tools for manipulating the producing organism developed, it becomes possible to alter biosynthetic genes in a specific manner in order to afford predictable changes in the chemical structure of the compound. Coupled to the knowledge on structure-activity relationship, such biosynthetic engineering becomes a powerful tool for improvement of pharmacological properties of natural products. This issue of “Current Topics in Medicinal Chemistry” is dedicated to the topic “Biosynthesis of Natural Products Applied to Drug Discovery”. The reviews from the leading scientists in the field of biosynthetic engineering presented in this issue highlight recent advances in applying this technology to the process of drug development. The reviews focus on biosynthesis of anti-bacterial, anti-fungal and anti-cancer antibiotics produced by actinomycetes, soil-dwelling bacteria with an enormous potential for production of diverse biologically active compounds. The first review from Baltz summarizes recent developments in understanding and engineering of the biosynthetic pathways for anti-bacterial lipopeptide antibiotics. These compounds penetrate the bacterial cell wall, leading to formation of pores, loss of electrical membrane potential and inhibition of peptidoglycan synthesis. The review not only compares biosynthetic pathways for several lipopeptides, but also suggests how this combined knowledge can be used for biosynthetic engineering. In particular, generation of new analogues of the clinically important antibiotic daptomycin is described. Several approaches for obtaining new daptomycin analogues are highlighted: module exchange in non-ribosomal peptide synthetases involved in daptomycin biosynthesis, heterologous transcomplementation, chemoenzymatic synthesis, and combinatorial biosynthesis. These studies provided important information on structure-activity relationship of lipopeptides, and will be extremely valuable for rational engineering of improved daptomycin analogues. In the next review, Caffrey et al. discuss issues of biosynthetic engineering of polyene macrolides, antibiotics currently used as anti-fungal and anti-parasitic agents. Polyene macrolides act through formation of hydrophilic channels in sterol-containing membranes, leading to leakage of ions and small molecules. The use of these antibiotics have been limited by serious side effects, and multiple attempts on generation of less toxic semi-synthetic derivatives have so far failed to bring any new polyene macrolides on the market.

Daptomycin is a clinically important antibiotic approved for the treatment of complicated skin and skin structure infections caused by Gram-positive pathogens, and for the treatment of bacteremia and endocarditis caused by Staphylococcus aureus. Daptomycin and related acidic cyclic lipopeptide antibiotics have ten amino acids in the ring, and exocyclic tails containing one or three amino acids. The N-termini of the exocyclic amino acids are generally coupled to long chain fatty acids. Biosynthesis is initiated by the coupling of fatty acids to the N-terminal amino acids, followed by the coupling of the remaining amino acids by nonribosomal peptide synthetase (NRPS) mechanisms, then cyclization and release of the lipopeptides. The biosynthetic genes for daptomycin, calcium dependent antibiotic (CDA), A54145 and friulimicin have been cloned, sequenced, analyzed bioinformatically, and in some cases genetically or biochemically. The information on the organization and expression of the NRPS and other genes has been exploited to generate combinatorial libraries of hybrid lipopeptide antibiotics related to daptomycin, including several compounds with very good antibacterial activities.

Polyene macrolides are potent antifungal agents that are also active against parasites, enveloped viruses and prion diseases. They are medically important as antifungal antibiotics but their therapeutic use is limited by serious side effects. In recent years there has been considerable progress in genetic analysis and manipulation of the streptomycetes that produce nystatin, amphotericin B, candicidin, pimaricin and rimocidin/CE-108-related polyenes. This has led to engineered biosynthesis of several new polyenes that are not easily obtained as semi-synthetic derivatives. This review summarises recent advances made since the subject was last reviewed in 2003. Polyene biosynthesis generally involves assembly and cyclisation of a polyketide chain, followed by oxidative modifications and glycosylation of the macrolactone ring. New derivatives have been obtained by engineering both early and late stages of polyene biosynthetic pathways. These compounds have allowed more detailed investigations of structure-activity relationships and some are likely to show improvements in therapeutic index. The biosynthetic approach is already yielding sufficient material for testing the toxicity and activity of new compounds, thus opening possibilities for discovery of leads for development of effective and safe antifungal and antiparasitic agents.

Glycopeptide antibiotics are complex natural products biosynthesized by several actinomycete genera. They inhibit bacterial growth by interfering with cell wall biosynthesis. Glycopeptide antibiotics consist of a heptapeptide skeleton highly modified through cross-links of the aromatic moieties. In addition, they are usually further embellished by chlorination, glycosylation, methylation, acylation and/or sulfation. The clinically used glycopeptides vancomycin and teicoplanin have become last resort antibiotics against multi-resistant Gram positive pathogens. In addition, secondgeneration glycopeptides with improved properties, obtained by semi-synthesis, have been developed. This has created considerable interest in augmenting the structural diversity of glycopeptides by complementing chemical methods, which are limited to few accessible positions, with biological means. The elucidation of the biosynthetic pathways leading to six different compounds in this class has thus expanded the toolbox for structural manipulations. We review the current understanding of glycopeptide biosynthesis, a requisite for producing additional derivatives. In recent years, several novel compounds have been obtained by mutasynthesis, genetic manipulation, chemoenzymatic approaches or a combination thereof. The potential of these methods for creating clinically valuable compounds will be discussed.

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin®). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. λ RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage φC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.

An increasing appreciation of carbohydrates as components of natural products has uncovered new opportunities in carbohydrate-based drug design. Glycosylated natural products produced by microorganisms contain a variety of different sugars. Usually the biosynthetic pathways to deoxysugars start from a monosacchride-1-phosphate which is converted to a NDP-hexose by a nucleotidyltransferase. Modification of this intermediate by different enzymes (e.g. dehydratases, epimerases, aminotransferases) yields the final sugar. In contrast to microorganisms, plant products mostly contain glucose, galactose, rhamnose and xylose as structural elements. In all organisms the nucleotide-activated sugar is attached to an aglycon by a glycosyltransferrase (GT). As no single universal GT has been uncovered yet, accomplishing the generation of novel glycosylated compounds requires a deep understanding of the function of glycosyltransferases (GTs) and its specificity. In this review we will present important drugs that contain sugar components. We will give an overview about the existing natural product GTs and we will discuss the structural features of GTs. Through specific examples within different compound classes, we will highlight recent examples of metabolic and combinatorial engineering approaches successfully applied to the production of novel glycosylated compounds.

Bioactive natural products are frequently glycosylated with saccharide chains of variable length. These sugars are important for the biological activity of the compounds and they contribute to the interaction with the biological target. The increasing knowledge of sugar biosynthesis pathways and the isolation of a large number of sugar gene clusters from antibiotic-producing actinomycetes are providing tools for combinatorial biosynthesis approaches that can generate potentially improved derivatives with altered sugars in their architecture. Novel derivatives of known bioactive natural products can be produced either in the producer organisms or in heterologous hosts by using different combinatorial biosynthesis strategies. In this article, recent advances in the field are discussed, illustrating the alternative approaches of gene inactivation, gene expression, combining gene inactivation and gene expression, co-expression of genes from different pathways or the use of sugar cassette plasmids to endow a host with the capability of synthesizing new sugars.

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