**5. Modification of natural scaffolds**

Natural resources are and will continue to provide structurally and mechanistically new molecules that serve as useful drugs or lead compounds. One should realize, however, that natural antibiotics rarely possess the appropriate characteristics to be directly considered as drugs. Instead, they typically need to undergo chemical modifications in order to be translated into functional drugs. The goal of such projects may be to improve the pharmacokinetic properties of drug leads (e.g. increase stability and bioavailability) or produce derivatives with higher activity and wider antibiotic spectrum (e.g. by incorporating moieties to evade bacterial efflux pumps or to engage into additional interactions with the bacterial target protein). Both focused rational design and combinatorial chemistry approaches backed up by structural studies of targets complexed with natural or synthetic antibacterials and their derivatives have resulted in numerous optimized antibiotic drugs (Brötz-Oesterhelt & Sass, 2010; Butler & Cooper, 2011; Newman & Cragg, 2007).

Combinatorial biosynthesis is a rapidly expanding field in natural antibiotic optimization (Baltz, 2008; Kopp & Marahiel, 2007). The modular nature of polyketide synthases and nonribosomal peptide synthetases enables generation of natural product variants by exchange or alteration of individual modules within the bioassembly line. Many polyketides and nonribosomal peptides are not amenable to chemical synthesis and semisynthetic modification due to extreme structural complexity. Here, chemoenzymatic approaches represent a viable alternative. One prominent example is the generation of a library of lipopeptides based on the daptomycin structure (Nguyen et al., 2006). The daptomycin biosynthetic pathway was engineered by module and subunit exchange, and inactivation of a tailoring enzyme. Some of the lipopeptide variants produced in fermentations were highly active antibiotics. Another group used error-prone PCR to generate gene mutants of glycosyltransferase that catalyses glucosylation of macrolide antibiotic oleandromycin (Williams et al., 2007). Thereby, they managed to broaden the specificities of the glycosyltransferase for acceptor substrates as well as donor nucleoside diphospho-sugars. Such innovative chemoenzymatic strategies combined with semisynthetic modification of natural products (novel and old) seem to provide a powerful tool for the development of new and improved antibiotics.

#### **6. References**

42 Antimicrobial Agents

isolation and laboratory cultivation of individual species" (Miao & Davies, 2009). At the heart of metagenomics lies the recovery and sequencing of genomes of entire microbial communities occupying diverse ecological niches. Thereby, even the uncultivable microorganisms are addressed. The gathered genetic information is then scanned for potential biosynthetic genes in the hope for identification of novel natural products in a similar way as previously discussed (see section 4.5) (Banik & Brady, 2010; Miao & Davies, 2009). Alternatively, metagenomic expression libraries can also be directly assayed for functional products (Brady, 2007). However, due to methodological obstacles no complex biosynthetic gene clusters have been recovered from environmental DNA (eDNA) to date

One of the biggest problems in metagenomics is the inefficient cloning of extremely large DNA segments needed to harbor intact gene clusters for preparation of metagenomic libraries. The transformation of vectors such as cosmids or bacterial artificial chromosomes to surrogate hosts is the main factor that limits construction of libraries with acceptable complexity. Moreover, the host might not efficiently express biosynthetic transgenes because of differences in codon usage or incompatibility of promoters (Miao & Davies, 2009; B.K. Singh & Macdonald, 2010). Finally, it is imperative to enrich microbial populations for strains with potential to produce complex secondary metabolites (see section 4.2) (Miao & Davies, 2009) or enrich isolated eDNA samples for genes of interest (Banik & Brady, 2010)

Natural resources are and will continue to provide structurally and mechanistically new molecules that serve as useful drugs or lead compounds. One should realize, however, that natural antibiotics rarely possess the appropriate characteristics to be directly considered as drugs. Instead, they typically need to undergo chemical modifications in order to be translated into functional drugs. The goal of such projects may be to improve the pharmacokinetic properties of drug leads (e.g. increase stability and bioavailability) or produce derivatives with higher activity and wider antibiotic spectrum (e.g. by incorporating moieties to evade bacterial efflux pumps or to engage into additional interactions with the bacterial target protein). Both focused rational design and combinatorial chemistry approaches backed up by structural studies of targets complexed with natural or synthetic antibacterials and their derivatives have resulted in numerous optimized antibiotic drugs (Brötz-Oesterhelt & Sass, 2010; Butler & Cooper, 2011; Newman

Combinatorial biosynthesis is a rapidly expanding field in natural antibiotic optimization (Baltz, 2008; Kopp & Marahiel, 2007). The modular nature of polyketide synthases and nonribosomal peptide synthetases enables generation of natural product variants by exchange or alteration of individual modules within the bioassembly line. Many polyketides and nonribosomal peptides are not amenable to chemical synthesis and semisynthetic modification due to extreme structural complexity. Here, chemoenzymatic approaches represent a viable alternative. One prominent example is the generation of a library of lipopeptides based on the daptomycin structure (Nguyen et al., 2006). The daptomycin biosynthetic pathway was engineered by module and subunit exchange, and inactivation of a tailoring enzyme. Some of the lipopeptide variants produced in fermentations were highly

before the metagenomic library is constructed to minimize background.

(Miao & Davies, 2009).

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**3** 

*Mexico* 

**Natural Antimicrobial** 

Renaud Condé, Martha Argüello,

*Instituto Nacional de Salud Pública* 

**Peptides from Eukaryotic Organisms** 

*Centro de Investigación Sobre Enfermedades Infecciosas,* 

Javier Izquierdo, Raúl Noguez, Miguel Moreno and Humberto Lanz

Antimicrobial peptides (AMP) are usually described as being short (less than 100 a.a.), gene encoded, ribosome synthesized, polypeptide substances that have antimicrobial activity. For simplicity reasons, we will exclude peptaibol and other non-ribosomaly synthetized

The first peptidic antibiotic was described in 1968 coming from the *Manduca sexta* and was of linear nature; since then the number of antimicrobial peptide discovered have grown asymptotically. Though loose homology has been found between certain set of antimicrobial peptides; it has proven difficult to classify the AMP through their primary structure. Antimicrobial peptides show a great diversity of primary structures, and their short size do not permit robust evolutionary classification, but for the most close related peptides. The primary structures signature of the different AMP families may have arisen independently, and in some case these structures homology are the result of convergent evolution rather than a common ancestry. Nevertheless in order to classify the new components, general classification methods have been established. So far this has been done regardless of evolutionary relationship, source or activity. The criteria that have been commonly used are the number of disulfide bridges and particular amino-acid composition. In 2005 P. Bullet and co-workers suggested a 3 categories classification namely: α-Helical host defense peptides (HDPs), β-Sheet HDPs, Flexible HDPs rich in certain amino acids (Bulet et al., 1999). Though most AMP would fit in this classification, little insight about function can be inferred from the class relation; nor does it give any comparative information between

More recently Tomas Ganz proposed a structural classification of the AMP based on their secondary structure (Ganz, 2003b). The classes proposed included antimicrobial peptides with 4 disulfide bridges with alpha helix and beta sheet mixed structures, 3 disulfide bridges with alpha helix and beta sheet mixed structures, 3 disulfide bridges with beta sheet motif, 3 disulfide bridges with two alpha helix and beta sheet mixed structures, 2 disulfide bridges with beta-sheet structures, one disulfide bridge cyclic peptide and alpha helical peptides.

The classification proposed here contains 9 different peptide structure families. The last group consider hybrid structure peptide possessing structural features of more than one AMP class.

**1. Introduction** 

antibiotic from our classification.

peptides belonging to the same class.

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