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Drug Discovery & Development

The Therapeutic Potential of Lantibiotics

By Steven Boakes Lantibiotics are a growing class of diverse bacterial peptides with a range and Sjoerd Wadman of of structures and functions. They offer real potential in combating bacterial Novacta Biosystems Ltd infections and – in an age when increasing numbers of commonly used antimicrobials are falling prey to bacterial resistance – they could form a significant addition to the therapeutic armamentarium.

Lantibiotics are bacterial peptides that are ribosomally that lantibiotics are more common than previously synthesised and post-translationally modified to their suspected, are produced by many different organisms and active form. To date, relatively few lantibiotics have been have a wide variety of biological activities and functions. isolated and characterised, but with recent advances in Structurally, the lantibiotics are characterised by the biotechnology, new members of the class are being presence of or methyllanthionine amino acids discovered at an accelerating rate. New discoveries suggest formed through the intramolecular cross-linking of cysteine thiols to dehydrated serine and threonine residues Figure 1: Lantibiotic structures. Modified residues have been coloured red. respectively. Within this diverse group of peptides, many Dha, Didehydroalanine; Dhb, didehydrobutyrine; and Abu, 2-aminobutyric acid compounds with remarkable thermal and metabolic stability exist, and the class may harbour many potential candidates for development as therapeutic agents. This article highlights some of the features of lantibiotics and provides an overview of the therapeutic potential of this compound class.

DIVERSITY AND BIOSYNTHESIS The lantibiotics are a diverse class of peptides produced by a range of Gram-positive bacteria (see Figure 1). Initially synthesised on the as unmodified peptides including a leader sequence, which is subsequently proteolytically removed, these prepeptides undergo a series of modifications prior to conversion to their active form and transport from the cell (see Figure 2). Modifications of the prepeptide include the dehydration of specific

Figure 2: Biosynthesis of mersacidin. The mrsA gene encodes the mersacidin serine and threonine residues; these residues then cross-link peptide. The C-terminal is first decarboxylated forming a precursor to the with cysteine thiols forming the characteristic aminovinyl-cysteine moiety. MrsM then catalyses dehydration of a serine and (methyl)lanthionine bridges which impart structural four threonine residues, and cyclisation of the molecule. The protein, MrsT, is then believed to assist in the subsequent proteolytic cleavage of the leader stability and a reduced susceptibility to proteolytic attack. sequence and export of mersacidin from the cell However, in some instances the dehydrated amino acids do not cross-link and are retained in the final structure of the molecule. Additional modifications have been described and include deamination of dehydrated N-terminal serine or threonine residues as in Pep5, oxidative decarboxylation of a C-terminal cysteine yielding an aminovinylcysteine (for example, mersacidin) and, more recently described, the presence of residues such as chlorotryptophan and hydroxyproline (1). The genes encoding the biosynthesis of lantibiotics are clustered (see Figure 3, page 24) and designated the generic locus symbol lan. To date, several lantibiotic gene clusters

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have been identified and sequenced. Although the represent an immediate problem. The cinnamycin group of arrangement and number of genes vary between clusters, lantibiotics, which include the duramycins, have been each contains: (i) a structural gene, lanA, which encodes for shown to bind to the cell membrane component, the prepeptide; (ii) a single lanM gene or otherwise a lanB phosphatidylethanolamine (PE), the substrate of and lanC, the products of which are responsible for the phopholipase A2 (5). introduction of crosslinks in the prepeptide; and (iii) a lanT Meanwhile the lantibiotic SapB represents an alternative (except for epicidin 280), responsible for the secretion of the class of lantibiotics, which do not bind to lipid II or PE. peptide from the cell. SapB produced by Streptomyces coelicolor is 21 residues long Other genes that are typically located amongst the and is an amphiphilic molecule, which acts as a cluster include genes, which encode for immunity and biosurfactant involved in supporting the aerial growth of regulation, as well as those responsible for specific processing Streptomyces mycelia (6). The diversity in the mode of functions. Studies have demonstrated that the lantibiotic action demonstrated by different lantibiotics reflects the biosynthetic machinery will accept a number of changes to structural variation in this class of molecules. Furthermore, the amino acid sequence and still produce fully processed as genomic studies are indicating that lantibiotics are more lantibiotics (2). This relaxed specificity – as well as the widespread than previously thought, new lantibiotics with ribosomal synthesis of the prepeptide (in contrast to multi- novel modes of action and potential therapeutic enzyme complexes) – make the lantibiotics particularly applications are likely to be uncovered. attractive to direct manipulation by targeted mutagenesis and/or combinatorial biosynthesis as a means of isolating APPLICATIONS lantibiotic variants with improved activity. At the time of writing, there are no lantibiotics approved for , arguably the best known lantibiotic, has been clinical use. Without doubt, this is due in part to the historic used as a food preservative for several decades and is difficulty of producing lantibiotics in high purity and currently used as such in over 40 countries, demonstrating quantity. However, recent advances in biotechnology have good activity against a wide range of bacteria. Its activity resulted in a considerable increase in our understanding of along with similar lantibioitics, such as subtilin, lies in its lantibiotic biosynthesis, and have opened up significant new ability to interact with the biosynthetic possibilities for structural optimisation and improvement in intermediate lipid II, forming pores in the cell membrane production protocols. Chemical synthesis and derivitisation which results in an efflux of metabolites from the cell and of lantibiotics is also a developing field, and represents disruption of vital ion gradients (3). Mersacidin and another approach towards the generation of therapeutically actagardine also bind to lipid II, but interact with an useful analogues. alternative part of the molecule to nisin, resulting in the As noted in the introduction, even the relatively small inhibition of transglycosylation and, ultimately, the number of lantibiotics characterised to date have a range of prevention of cell wall biosynthesis (4). Both compounds biological effects. Most prominent amongst these is potent demonstrate potent activity against Gram-positive antibacterial activity against many organisms, including Figure 3: Lantibiotic biosynthetic gene pathogens and – crucially – vancomycin-resistant strains are problematic pathogens like MRSA, VRE and Clostridium organisation susceptible, indicating that cross-resistance should not difficile. In an age when increasing numbers of commonly used antimicrobials fall prey to bacterial resistance, it seems most likely that in the immediate future the most sustained efforts to harness the therapeutic potential of lantibiotics will concentrate on their use as antibacterials. Lantibiotics are generally poorly absorbed via the oral, transdermal or pulmonary routes and most applications under investigation focus on topical or gastro-intestinal use. Thus mutacin 1140, a lantibiotic produced by Streptococcus mutans, is currently in preclinical development as a treatment for streptococcal throat infections and as a prophylactic treatment for dental caries (7). Following a probiotic approach, lozenges containing Streptococcus salivarius that produces the lantibiotic salivaricin A, are marketed as an oral care product. Nisin has been suggested for a number of applications, including superficial skin infections and for the treatment of gastric ulcers. It was investigated for the treatment of

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Clostridium difficile Associated Diarrhoea (CDAD), and its antibacterial and sporicidal properties appear most suitable Steven Boakes is a Senior Scientist at Novacta Biosystems. He received his PhD in Molecular Biology for this application. However, rapid degradation of nisin by in 2002 from the University of Cambridge under the intestinal enzymes makes delivery to the site of infection supervision of Professor Peter Leadlay. Following a (the colon) problematic. Type B lantibiotics, such as post-doctoral position with the Gene Expression and mersacidin and actagardine, are more extensively cyclised Protein Biochemistry group at GSK, he joined Novacta and stable to proteolysis, thus representing more promising in 2003 where his work has focused on the study and manipulation of lantibiotic biosynthetic gene clusters. starting points for delivery to the colon. A chemically Email: [email protected] modified Class II lantibiotic with selective activity against Clostridium difficile, NVB302, shows considerable promise Sjoerd Wadman is Head of Chemistry at Novacta as a treatment for CDAD and is in preclinical development. Biosystems. He completed his PhD in Synthetic The thermal stability of lantibiotics facilitates their use in Organic Chemistry at the University of Southampton (UK) and since then worked as a team and project a variety of medical applications (8). Gallidermin and other leader on a multitude of medicinal chemical projects lantibiotics can be incorporated into prosthetic joint cement at GlaxoSmithKline, Morphochem and Novacta. His and are being evaluated as prophylactics against implant- present focus is on the utilisation of and associated infections. Preliminary tests have also lantibiotics as therapeutically useful medicines. demonstrated that both gallidermin and epidermin are Email: [email protected] active against Propionibacterium acne, one of the causative agents in acne and as such could potentially be utilised in perhaps one of the most promising features of this class of the treatment of such diseases. compounds is their favourable resistance profiles against A further group of Type B lantibiotics comprising Gram-positive pathogens and, as such, current cinnamycin, duramycin and ancovenin have only weak development is likely to focus heavily on the use of antibacterial activity but are potent inhibitors of human lantibiotics as antibacterials. enzymes, notably phospholipase A2 and angiotensin converting enzyme. Most advanced in clinical development References is duramycin, undergoing Phase II clinical trials for the 1. Castiglione F et al, Determining the structure and mode treatment of reduced mucociliary clearance in cystic fibrosis of action of microbisporicin, a potent lantibiotic active and Phase II trials for treating dry eye syndrome (9). against multiresistant pathogens, Chem & Biol 15, Systemic use of lantibiotics, for instance as pp22-31, 2008 antimicrobials, requires more stringent pharmacodynamic 2. Dawson MJ, The lantibiotics: an underexploited class of and pharmacokinetic demands to be met. Given their natural products with broad potential, Chemistry Today structural diversity, lantibiotics are bound to display a range 24(6), pp25-27, 2006 of metabolic susceptibilities, most notably towards 3. Lubelski J et al, Biosynthesis, immunity, regulation, proteolytic cleavage. The protein binding, distribution, mode of action and engineering of the model lantibiotic excretion and toxicity of intravenously delivered lantibiotics nisin, Cell Mol Life Sci 65, pp455-476, 2008 has received scant attention to date, and it is likely therefore 4. Brötz H et al, The lantibiotic mersacidin inhibits that the development as intravenously delivered peptidoglycan synthesis by targeting lipid II, Antimicrob therapeutics will require a more sophisticated approach that Agents Chemother 42(1), pp154-160, 1998 will include structural modifications via biochemical or 5. Iwamoto K et al, Curvature-dependent recognition chemical means. of ethanolamine phosopholipids by duramycin and cinnamycin, Biophys J 93, pp1,608-1,619, 2007 FUTURE PROSPECTS 6. Capstick DS et al, SapB and the chaplins: connections Although no lantibiotics are currently in clinical use, they between morphogenetic proteins in Streptomyces represent an intriguing class of compounds with potential coelicolor, Mol Micro 64(3), pp602-613, 2007 applications in a diverse range of therapeutic areas. The 7. Smith L and Hillman JD, Therapeutic potential of type depth of this diversity is likely to increase in the coming A (I) lantibiotics, a group of cationic peptide , years as more lantibiotics are discovered and advances are Curr Opin Microbiol 11, pp401-408, 2008 made in the understanding of the biosynthetic machinery. 8. Hancock REW and Sahl H-G, Antimicrobial In vitro studies of individual enzymatic components are and host-defense peptides as new anti-infective providing an insight into the enzymology at the molecular therapeutic strategies, Nature Biotechnology 24(12), level, and could help in the development of more pp1,551-1,557, 2006 efficacious lantibiotic variants and further broadening of 9. Grasemann H et al, Inhalation of Moli1901 in patients therapeutic targets through enzyme evolution. Meanwhile, with cystic fibrosis, Chest 131, pp1,461-1,466, 2007

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