Current Topics in Medicinal Chemistry

ISSN: 1568-0266

Current Topics in Medicinal Chemistry
Volume 8, Number 8, 2008

Contents

Biosynthesis of Natural Products Applied to Drug Discovery
Guest Editor: Sergey B. Zotchev


Editorial Pp. 616-617


Biosynthesis and Genetic Engineering of Lipopeptide Antibiotics Related to Daptomycin Pp. 618-638
Richard H. Baltz
[Abstract]


Biosynthetic Engineering of Polyene Macrolides Towards Generation of Improved Antifungal and Antiparasitic Agents Pp. 639-653
Patrick Caffrey, Jesus F. Aparicio, Francisco Malpartida and Sergey B. Zotchev
[Abstract]


Biosynthesis of Glycopeptides: Prospects for Improved Antibacterials Pp. 654-666
Stefano Donadio and Margherita Sosio
[Abstract]


Combinatorial Biosynthesis, Metabolic Engineering and Mutasynthesis for the Generation of New Aminocoumarin Antibiotics Pp. 667-679
L. Heide, B. Gust, C. Anderle and S.-M. Li
[Abstract]


Glycosyltransferases, Important Tools for Drug Design Pp. 680-709
Andriy Luzhetskyy, Carmen Méndez, Jose A. Salas and Andreas Bechthold
[Abstract]


Deoxysugars in Bioactive Natural Products: Development of Novel Derivatives by Altering the Sugar Pattern Pp. 710-724
Carmen Méndez, Andriy Luzhetskyy, Andreas Bechthold and José A. Salas
[Abstract]


Molecule of Month Pp. 725




Abstracts

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Editorial

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. The review describes cloning and analysis of biosynthetic genes for production of pimaricin, nystatin, amphotericin B, candicidin/FR008 and rimocidin by several research groups. The approach of biosynthetic engineering of these polyene macrolides has yielded several new analogues with retained anti-fungal activity and substantially reduced in vitro toxicity that might become new scaffolds for the development of safer anti-fungal drugs.

The third review by Donadio and Sosio focuses on biosynthesis of glycopeptide antibiotics. Vancomycin and teicoplanin, representatives of this group of compounds, are currently the last line of defence in treatment of bacterial infections caused by multi-resistant pathogens. These antibiotics inhibit cell wall biosynthesis in bacteria by blocking formation of a peptidoglycan. The review describes how biosynthetic engineering of glycopeptide antibiotics can complement synthetic chemistry in generation of new antibiotic analogues. Once the biosynthetic routes for glycopeptides have been established, new analogues were obtained by genetic manipulation of the producing strains, mutasynthesis, and chemoenzymatic approach. Especially promising seem to be modifications of the core cyclic peptide structures by attachment of new moieties, in particular novel sugar residues.

The review by Heide et al. presents recent developments in biosynthetic engineering of aminocoumarins, antibiotics used against bacterial infections and also having potential in anti-cancer therapy. Aminocoumarins are potent inhibitors of bacterial gyrase and topoisomerase IV, and were also shown to modulate cytotoxic activity of several anti-cancer agents. Heide et al. describe cloning, analysis, heterologous expression and manipulation of genes for biosynthesis of three aminocoumarin antibiotics. In addition to the genetic manipulation, new aminocoumarins have been afforded through mutasynthesis and metabolic engineering of the producing organisms. Almost 100 new aminocoumarin analogues have successfully been generated, and many limitations were overcome through innovative approaches of modifying precursor supply and regulatory cascades in the producing organisms.

The next review, presented by Luzhetskyy et al., deals with glycosyltransferases, enzymes performing decoration of many natural products with sugar moieties. These moieties are found in natural products displaying a wide range of biological activities, including anti-bacterial, anti-fungal and anti-cancer, and believed to be crucial for interaction with cellular targets. The review highlights recent developments in understanding the specificity of glycosyltransferases from plants and microorganisms towards both recipient aglycone and sugars. This is being achieved through fundamental studies on the structures and catalytic mechanisms of these enzymes. Finally, the authors describe how this knowledge is being successfully used for engineering of glycosyltransferases and generation of new compounds with altered glycosylation patterns both in vitro and in vivo.

The final review by Mendes et al. is also concerned with glycosylation of natural products, but is focused on supply of different sugar moieties for this process. In the beginning, the review describes biosynthetic pathways for different sugars in antibiotic-producing actinomycete bacteria. Later on, the authors provide excellent examples of combinatorial biosynthesis designed to diversify natural products by means of alternative glycosylations. The latter is achieved by inactivation of specific sugar biosynthesis genes and expression of sugar pathways using “sugar cassettes”, which can be custom-engineered and introduced into different hosts in order to provide new sugar moieties. Using these approaches, novel glycosylated erythromycins, mithramycins, urdamycins, pikromycins, chlorobiocins, tetracenomycins and staurosporines have been generated. The latter suggests that these combinatorial tools are very useful for diversification of natural products in order to change properties that might be important for drug development.

I hope you will enjoy reading this issue, and may acquire new ideas based on the most interesting data reviewed here. I wish to thank Dr Allen B. Reitz for the invitation to compose and edit this issue. I would also like to thank all authors for their valuable contributions, and the reviewers for being rigorous in their evaluation of the manuscripts.


Sergey B. Zotchev
Department of Biotechnology,
Norwegian University of Science and Technology,
S. Saelands v 6/8, 7491 Trondheim,
Norway


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Biosynthesis and Genetic Engineering of Lipopeptide Antibiotics Related to Daptomycin
Richard H. Baltz

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.


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Biosynthetic Engineering of Polyene Macrolides Towards Generation of Improved Antifungal and Antiparasitic Agents
Patrick Caffrey, Jesus F. Aparicio, Francisco Malpartida and Sergey B. Zotchev

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.


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Biosynthesis of Glycopeptides: Prospects for Improved Antibacterials
Stefano Donadio and Margherita Sosio

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, second-generation 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.


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Combinatorial Biosynthesis, Metabolic Engineering and Mutasynthesis for the Generation of New Aminocoumarin Antibiotics
L. Heide, B. Gust, C. Anderle and S.-M. Li

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.


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Glycosyltransferases, Important Tools for Drug Design
Andriy Luzhetskyy, Carmen Méndez, Jose A. Salas and Andreas Bechthold

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.


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Deoxysugars in Bioactive Natural Products: Development of Novel Derivatives by Altering the Sugar Pattern
Carmen Méndez, Andriy Luzhetskyy, Andreas Bechthold and José A. Salas

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.