**1. Introduction**

24 Antimicrobial Agents

Sarac, N. & Ugur, A. (2007). Antimicrobial activities and usage in folkloric medicine of some

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Weckesser, S., Engel, K., Simon-Haarhaus, B., Wittmer, A., Pelz, K. & Schempp, C.M. (2007).

Youn, H.J., Lakritz, J., Rottinghaus, G.E., Seo, H.S., Kim, D.Y., Cho, M.H. & March, A.E.

Zidorn, C. (2008). Sesquiterpene lactones and their precursors as chemosystematic markers

Zuo, G.Y., Wang, G.C., Zhao, Y.B., Xu, G.L., Hao, X.Y., Han, J. & Zhao, Q. (2008). Screening

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of Chinese medicinal plants for inhibition against clinical isolates of methicillinresistant *Staphylococcus aureus* (MRSA). *Journal of Ethnopharmacology*, Vol. 120, No. 2, Few drugs have made such a profound impact on modern medicine as antibiotics. With the discovery of sulfonamides, β-lactams, and subsequent antibiotic classes after World War II, bacterial infections with often fatal outcomes literally became curable overnight. These "magic bullets," however, suffer from a serious drawback; the use (and misuse) of antibiotics induces selection pressure resulting in the development of resistance traits in bacterial populations. The process is augmented by short generation times of bacteria enabling rapid mutation and selection of resistant strains, and a horizontal transfer of resistance genes. Bacterial pathogens resistant to more than one, or even most clinically used antibiotics, have become common (Fischbach & Walsh, 2009). Faced with the fact that a renewed pre-antibiotic era might be just around the corner, the World Health Day 2011 campaign "Antimicrobial resistance and its global spread" launched by the WHO offers a strategy to safeguard existing antibiotics for future generations, and contain the spread of antimicrobial resistance (World Health Organization, 2011). However, taking a more sensible approach in prescribing and using available antibiotic drugs will only help to put off the inevitable. In the battle against the ever-increasing multidrug resistance of pathogenic bacteria, we urgently need new alternatives to the currently available broadspectrum antibiotics. Here we review some current trends in antibiotic discovery focusing on the screening of natural products.

This chapter is composed of four parts. We start by briefly reviewing the history of antibiotic discovery, gradually moving from its most fruitful era in the 1940's and 50's to the unexpected outcome decline in the genomic era. Next, we address two fundamental questions of antibiotic research in the post-genomic age; namely, *where* to look for novel antibiotics and *how*. Finally, we conclude with a concise discussion on the modification of natural scaffolds to be translated into functional drugs.

#### **2. History of antibiotic discovery**

#### **2.1 The golden age of antibiotic discovery**

Before the discovery of prontosil, the forerunner of sulfonamide chemotherapeutics, the only available measures in combating bacterial infections, apart from practicing proper

Future Antibiotic Agents: Turning to Nature for Inspiration 27

Fig. 1. Timeline for the introduction of major, broad-spectrum antibiotic classes for systemic application in the clinic, and documented occurrence of bacterial resistance (adapted from (Brötz-Oesterhelt & Sass, 2010)). The asterisk denotes two new antibiotic classes with single representatives (i.e., lipopeptide daptomycin, and pleuromutulin retapamulin), both of which are intended for topical application. However, analogues for systemic application (oral or *i.v.*) are being developed (also see section 3.1.4). ESBL – extended spectrum β-lactamase, VISA – vancomycin intermediately resistant *Staphylococcus aureus*,

VRE – vancomycin-resistant *Enterococcus*, VRSA – vancomycin highly resistant *S. aureus*.

**2.2 The disappointing investment in combinatorial chemistry and high-throughput** 

Rediscovery of known antibiotics from screening microbial extracts and the development of highly effective synthetic (fluoro)quinolones caused a shift in antimicrobial drug R&D strategy in the industry. The search for antibiotics occurring in the environment was mostly abandoned (although semisynthetic modification of natural scaffolds resulted in numerous improved antibiotics (Fischbach & Walsh, 2009)) and the screening efforts were once again invested in synthetic compounds. Since the early 1990's combinatorial chemistry was employed to produce large libraries of compounds that demanded high-throughput assaying for activity. The advent of genomics and bioinformatics raised the hopes for identification of entirely new antibacterial targets by mining bacterial genomes. Genes conserved among bacteria but sharing no homology to eukaryotic counterparts were considered as potential targets, and their activity or expression were manipulated by mutation studies or knockout technology to examine whether their products were indispensable for bacterial survival *in vitro*. Following a functional analysis, selected targets were expressed using recombinant technology and purified. This allowed for setting up miniaturized inhibitory activity assays for screening vast

**screening in antibiotic discovery** 

hygiene, were vaccination and passive immunization. Although these approaches are still invaluable today, the advent of broad-acting antibacterial agents enabled rapid treatment of patients with infections even when the exact causative bacterial pathogen was unknown. Prontosil, later found to be a prodrug releasing folate antimetabolite sulfanilamide upon reduction *in vivo*, was a result of a screening campaign at Bayer, Germany in the early 1930's, aimed at finding synthetic dyes for the potential effect on hemolytic streptococcal infection (Greenwood, 2003). Although the premise that dyes in general should exert antibacterial activity turned out to be incorrect, prontosil paved the way for antimicrobial drugs, becoming the first commercially available antibacterial agent and remained in clinical use for 30 years. Moreover, it inspired new generations of sulfonamide chemotherapeutics, some of which remain on the market today. All major antibiotic classes that form pillars of antibacterial therapy were derived from natural sources − mostly microbial secondary metabolites (Molinari, 2009) − with the exception of sulfonamides and quinolones, inhibitors of bacterial DNA gyrase that were discovered in the 1960's.

The groundbreaking work of Rene Dubois, who studied antibiosis in pairs of soil microorganisms, eventually leading to the discovery of a mixture of peptidic antibiotics collectively termed tyrothricin (its component gramicidin is still in limited use), inspired Selman Waksman and Boyd Woodruff to adopt the principle in systematic search for novel antibiotics (Kresge et al., 2004). It is now well recognized that many microbes produce structurally extremely diverse, small molecules that are involved in complex intra- and interspecies signaling (Shank & Kolter, 2009). Antibiosis is only one of the many possible outcomes of such interaction, but is most readily detected: Waksman and Woodruff looked for growth inhibition zones surrounding single colonies of soil microorganisms cultured under different conditions and then isolated the active substance from pure cultures by activity-guided fractionation (Waksman & Woodruff, 1940). The same route led to the earlier serendipitous discovery of penicillin by Alexander Fleming in 1929 (Fleming, 1929).

It took almost 15 years to scale up penicillin production and demonstrate its efficacy and safety. By that time numerous other antibiotic types were emerging (Fig. 1). The major source of antimicrobials turned out to be soil actinomycetes, such as the *Streptomyces* species, and various fungi. Considering the abundance of soil microbes (there are estimates of 109-1010 bacteria in a single gram of soil belonging to some 104 operational taxonomic units (Curtis et al., 2006; Gans et al., 2005)) the explosion in antibiotic discovery that began in the early 1940's is not surprising from current perspective. However, not all species are equally represented. Moreover, many related bacteria or fungi produce the same or similar secondary metabolites. For example, streptothricin was found in ~10%, streptomycin in ~1%, and tetracycline and actinomycin in ~0.1% of randomly collected soil actinomycetes (Baltz, 2007). So when the most abundant antibiotics were identified the pace of natural antibiotic discovery gradually slowed down (Baltz, 2007, 2008), finally culminating in a 30 year-gap in launching an antibiotic with a novel scaffold to the market (Fig. 1). The situation was exacerbated by ever stricter regulatory demands on safety and efficacy of drugs, the wrongful perception that bacterial infections no longer posed a severe threat to human health, as well as the unfavorable economics of antibacterial development (revenue from antibiotics is significantly lower compared to drugs indicated for chronic diseases), leading to a withdrawal of Big Pharma from the antibiotic business (Brötz-Oesterhelt & Sass, 2010; Fischbach & Walsh, 2009; Projan, 2003). Meanwhile, the development and spread of bacterial resistance was steadily incising into our antibacterial arsenal (Fig. 1).

hygiene, were vaccination and passive immunization. Although these approaches are still invaluable today, the advent of broad-acting antibacterial agents enabled rapid treatment of patients with infections even when the exact causative bacterial pathogen was unknown. Prontosil, later found to be a prodrug releasing folate antimetabolite sulfanilamide upon reduction *in vivo*, was a result of a screening campaign at Bayer, Germany in the early 1930's, aimed at finding synthetic dyes for the potential effect on hemolytic streptococcal infection (Greenwood, 2003). Although the premise that dyes in general should exert antibacterial activity turned out to be incorrect, prontosil paved the way for antimicrobial drugs, becoming the first commercially available antibacterial agent and remained in clinical use for 30 years. Moreover, it inspired new generations of sulfonamide chemotherapeutics, some of which remain on the market today. All major antibiotic classes that form pillars of antibacterial therapy were derived from natural sources − mostly microbial secondary metabolites (Molinari, 2009) − with the exception of sulfonamides and quinolones, inhibitors

The groundbreaking work of Rene Dubois, who studied antibiosis in pairs of soil microorganisms, eventually leading to the discovery of a mixture of peptidic antibiotics collectively termed tyrothricin (its component gramicidin is still in limited use), inspired Selman Waksman and Boyd Woodruff to adopt the principle in systematic search for novel antibiotics (Kresge et al., 2004). It is now well recognized that many microbes produce structurally extremely diverse, small molecules that are involved in complex intra- and interspecies signaling (Shank & Kolter, 2009). Antibiosis is only one of the many possible outcomes of such interaction, but is most readily detected: Waksman and Woodruff looked for growth inhibition zones surrounding single colonies of soil microorganisms cultured under different conditions and then isolated the active substance from pure cultures by activity-guided fractionation (Waksman & Woodruff, 1940). The same route led to the earlier serendipitous discovery of penicillin by Alexander Fleming in 1929 (Fleming, 1929). It took almost 15 years to scale up penicillin production and demonstrate its efficacy and safety. By that time numerous other antibiotic types were emerging (Fig. 1). The major source of antimicrobials turned out to be soil actinomycetes, such as the *Streptomyces* species, and various fungi. Considering the abundance of soil microbes (there are estimates of 109-1010 bacteria in a single gram of soil belonging to some 104 operational taxonomic units (Curtis et al., 2006; Gans et al., 2005)) the explosion in antibiotic discovery that began in the early 1940's is not surprising from current perspective. However, not all species are equally represented. Moreover, many related bacteria or fungi produce the same or similar secondary metabolites. For example, streptothricin was found in ~10%, streptomycin in ~1%, and tetracycline and actinomycin in ~0.1% of randomly collected soil actinomycetes (Baltz, 2007). So when the most abundant antibiotics were identified the pace of natural antibiotic discovery gradually slowed down (Baltz, 2007, 2008), finally culminating in a 30 year-gap in launching an antibiotic with a novel scaffold to the market (Fig. 1). The situation was exacerbated by ever stricter regulatory demands on safety and efficacy of drugs, the wrongful perception that bacterial infections no longer posed a severe threat to human health, as well as the unfavorable economics of antibacterial development (revenue from antibiotics is significantly lower compared to drugs indicated for chronic diseases), leading to a withdrawal of Big Pharma from the antibiotic business (Brötz-Oesterhelt & Sass, 2010; Fischbach & Walsh, 2009; Projan, 2003). Meanwhile, the development and spread of

bacterial resistance was steadily incising into our antibacterial arsenal (Fig. 1).

of bacterial DNA gyrase that were discovered in the 1960's.

Fig. 1. Timeline for the introduction of major, broad-spectrum antibiotic classes for systemic application in the clinic, and documented occurrence of bacterial resistance (adapted from (Brötz-Oesterhelt & Sass, 2010)). The asterisk denotes two new antibiotic classes with single representatives (i.e., lipopeptide daptomycin, and pleuromutulin retapamulin), both of which are intended for topical application. However, analogues for systemic application (oral or *i.v.*) are being developed (also see section 3.1.4). ESBL – extended spectrum β-lactamase, VISA – vancomycin intermediately resistant *Staphylococcus aureus*, VRE – vancomycin-resistant *Enterococcus*, VRSA – vancomycin highly resistant *S. aureus*.

#### **2.2 The disappointing investment in combinatorial chemistry and high-throughput screening in antibiotic discovery**

Rediscovery of known antibiotics from screening microbial extracts and the development of highly effective synthetic (fluoro)quinolones caused a shift in antimicrobial drug R&D strategy in the industry. The search for antibiotics occurring in the environment was mostly abandoned (although semisynthetic modification of natural scaffolds resulted in numerous improved antibiotics (Fischbach & Walsh, 2009)) and the screening efforts were once again invested in synthetic compounds. Since the early 1990's combinatorial chemistry was employed to produce large libraries of compounds that demanded high-throughput assaying for activity. The advent of genomics and bioinformatics raised the hopes for identification of entirely new antibacterial targets by mining bacterial genomes. Genes conserved among bacteria but sharing no homology to eukaryotic counterparts were considered as potential targets, and their activity or expression were manipulated by mutation studies or knockout technology to examine whether their products were indispensable for bacterial survival *in vitro*. Following a functional analysis, selected targets were expressed using recombinant technology and purified. This allowed for setting up miniaturized inhibitory activity assays for screening vast

Future Antibiotic Agents: Turning to Nature for Inspiration 29

is a problem accessing it. A majority (maybe up to 99%) of microbes, renowned for their rich and diverse metabolism, cannot be cultured in a laboratory, at least not under standard conditions (Amann et al., 1995; Li & Vederas, 2009). There are species of microbes that thrive in geographical or ecological niches, such as deep sea and thermal springs, or as symbionts of plants and animals, respectively, that still await to be explored. Besides rediscovery, a major obstacle that can impede natural product research is that some compounds are found in the environment in rather low concentrations, complicating their detection and isolation in

Nevertheless, the thesis that the laborious screening for natural products with antibiotic activity is still worth the effort is supported by several facts. The parvome displays structural diversity unmatched by synthetic compounds; secondary metabolites often possess numerous chiral centers and display astonishing steric complexity. Furthermore, many natural antibiotics display complex and multilayer mechanisms of action that might not have been devised by rational design. Last but not least, millions of years of evolution have optimized antibiotics with respect to affinity and specificity for their targets, as well as physicochemical properties to penetrate bacterial envelopes (Butler & Buss, 2006; Pelaez, 2006; Swinney & Anthony, 2011). Encouragingly, owing to the revival of screening for natural antimicrobials or reinspection of collections of old antibiotics in the last decade, we have witnessed attempts to develop antibiotics based on novel chemicals templates, such as lipopeptides, pleuromutilins, ramoplanins, and actinonins (Butler & Buss, 2006; Butler & Cooper, 2011). Drugs based on new scaffolds exerting novel mechanisms of action should be superior to existing antibiotic classes in the fight against multi-drug resistant pathogens (Butler & Buss, 2006). Of note, two such antibiotics have recently been approved for use in humans. Daptomycin, the first member of lipopeptide antibiotics, acts through a complex mechanism involving the disruption of the bacterial membrane leading to inhibition of DNA, RNA, and protein synthesis, and is indicated for the treatment of skin and skin structure infections caused by Gram-positive pathogens (Baltz et al., 2005). Retapamulin, a pleuromutilin type antibiotic with indications similar to those of daptomycin, selectively inhibits the P site of peptidyl transferase centre on the bacterial 50S ribosomal subunit, exhibiting a mechanism that differs from other protein synthesis-inhibiting antibiotics

It is important to realize that all small molecular weight microbial products are active even though they might not induce antibiosis at concentrations found in the environment, suggesting their role as signaling molecules (Dufour & Rao, 2011; Miao & Davies, 2010; Shank & Kolter, 2009; Wyatt et al., 2010). Remarkably, this holds true even for well established antibiotics; a number of recent studies reported specific modulation of gene expression in different bacteria when exposed to subinhibitory concentrations of various antibiotics (Davies et al., 2006; Fajardo & Martinez, 2008; Linares et al., 2006). Reevaluation of known natural products for traits other than antibiosis might thus present another route leading to antibacterial drug discovery; inhibiting the production of metabolites that provide the producing microbe with an advantage in colonizing a certain niche could prove

The search for new antibiotics compounds goes hand in hand with the discovery of new (micro)organisms producing them. For this purpose the search has continued on land and at

to be a fruitful approach in designing antimicrobials (Wyatt et al., 2010).

quantities allowing structural and functional studies.

(Dubois & Cohen, 2010; Schlunzen et al., 2004).

**3. Where do we search for natural antibiotics?** 

chemical libraries against isolated targets, as well as elucidation of targets' structures to guide the subsequent optimization of leads. Despite enormous efforts in the last 20 years, however, the target-oriented drug discovery approach has not resulted in a single new antimicrobial chemotherapeutic. The reasons for that are multitude (reviewed in detail in (Baltz, 2006; Brötz-Oesterhelt & Sass, 2010; Projan, 2003)).

Firstly, the abandonment of whole-cell assays meant that cell penetrating capabilities were not a selection criterion for hits early in the discovery process. Therefore, most compounds that were highly active against an isolated target possessed no antimicrobial activity. Secondly, it appears that the properties of antibiotics in general do not conform to Lipinski's rule (Lipinski et al., 2001); they are more polar and have a higher molecular weight than drugs for other indications (O'Shea & Moser, 2008). Chemical libraries, on the other hand, had mostly been designed to meet Lipinski's criteria, and were thus likely biased against antibiotic compounds (Payne et al., 2007). Thirdly, inhibiting targets that were validated to be indispensible for bacterial survival *in vitro* does not necessarily lead to antibacterial effect *in vivo*. A prominent example is that of the type II fatty acid synthesis (FASII) pathway, intrinsic to bacteria: bacterial pathogens susceptible to FASII inhibitors *in vitro* were shown to be resistant to them when cultured in the presence of unsaturated fatty acids, or *in vivo* upon infection of rodents (Brinster et al., 2009). This indicates that bacteria can thrive in the nutrient-rich environment of the host by acquiring exogenous fatty acids, fully bypassing FASII pathway inhibition. Similarly, there is no guaranty that a target essential for viability of one bacterial strain will also be indispensible in others − as alternative biochemical pathways may be present that allow the targeted pathway to be circumvented (Gentry et al., 2003).

#### **2.3 Reappraising the natural products**

Historically, most drugs were derived from natural products. This trend continues today with ~50% of new small molecule drugs approved between the years 1981 and 2006 being either (semi)synthetic derivatives of compounds isolated from natural sources or synthetic mimetics of pharmacophores found in natural products (Newman & Cragg, 2007). In the field of antibacterial drugs, the trend is even more pronounced. Of 98 new molecular entities that were approved for human therapy in the same time period, only 23 are of totally synthetic origin, most of them (20) belonging to the quinolone group (Newman & Cragg, 2007). A notable exception is linezolid, the first and, to date, the only representative of oxazolidinone chemotherapeutics developed from initial hits of *cell-based* screening efforts for antibacterial activity from a chemical library (Barbachyn & Ford, 2003; Slee et al., 1987). Among the 40 antibacterial compounds currently undergoing clinical trials, 20 are natural product-derived, 18 are synthetic, and 2 are of unknown origin (Butler & Cooper, 2011). Interestingly, while the ratio of natural product-derived vs. synthetic entities is roughly 1:1 in phases I and II, the former predominate in phase III (i.e., 4:1). Moreover, there are more novel antibacterial classes among natural-product derived antibiotics compared to synthetic ones (a total of 7 new chemical scaffolds vs. 4) in the pipeline.

Disappointment from antibacterial drug discovery in the genomic era brought a renewed interest in screening natural products (Baltz, 2008; Davies, 2011; Li & Vederas, 2009; Molinari, 2009). Chemists have been isolating and analyzing secondary metabolites from plants, fungi, and bacteria for over 200 years, yet only a small percentage of species has been addressed (Li & Vederas, 2009). Undoubtedly, the natural supply of small molecules (sometimes referred to as parvome (Davies, 2011), from the Latin *parvus* meaning small) remains vast; however, there

chemical libraries against isolated targets, as well as elucidation of targets' structures to guide the subsequent optimization of leads. Despite enormous efforts in the last 20 years, however, the target-oriented drug discovery approach has not resulted in a single new antimicrobial chemotherapeutic. The reasons for that are multitude (reviewed in detail in (Baltz, 2006; Brötz-

Firstly, the abandonment of whole-cell assays meant that cell penetrating capabilities were not a selection criterion for hits early in the discovery process. Therefore, most compounds that were highly active against an isolated target possessed no antimicrobial activity. Secondly, it appears that the properties of antibiotics in general do not conform to Lipinski's rule (Lipinski et al., 2001); they are more polar and have a higher molecular weight than drugs for other indications (O'Shea & Moser, 2008). Chemical libraries, on the other hand, had mostly been designed to meet Lipinski's criteria, and were thus likely biased against antibiotic compounds (Payne et al., 2007). Thirdly, inhibiting targets that were validated to be indispensible for bacterial survival *in vitro* does not necessarily lead to antibacterial effect *in vivo*. A prominent example is that of the type II fatty acid synthesis (FASII) pathway, intrinsic to bacteria: bacterial pathogens susceptible to FASII inhibitors *in vitro* were shown to be resistant to them when cultured in the presence of unsaturated fatty acids, or *in vivo* upon infection of rodents (Brinster et al., 2009). This indicates that bacteria can thrive in the nutrient-rich environment of the host by acquiring exogenous fatty acids, fully bypassing FASII pathway inhibition. Similarly, there is no guaranty that a target essential for viability of one bacterial strain will also be indispensible in others − as alternative biochemical pathways may be present that

Historically, most drugs were derived from natural products. This trend continues today with ~50% of new small molecule drugs approved between the years 1981 and 2006 being either (semi)synthetic derivatives of compounds isolated from natural sources or synthetic mimetics of pharmacophores found in natural products (Newman & Cragg, 2007). In the field of antibacterial drugs, the trend is even more pronounced. Of 98 new molecular entities that were approved for human therapy in the same time period, only 23 are of totally synthetic origin, most of them (20) belonging to the quinolone group (Newman & Cragg, 2007). A notable exception is linezolid, the first and, to date, the only representative of oxazolidinone chemotherapeutics developed from initial hits of *cell-based* screening efforts for antibacterial activity from a chemical library (Barbachyn & Ford, 2003; Slee et al., 1987). Among the 40 antibacterial compounds currently undergoing clinical trials, 20 are natural product-derived, 18 are synthetic, and 2 are of unknown origin (Butler & Cooper, 2011). Interestingly, while the ratio of natural product-derived vs. synthetic entities is roughly 1:1 in phases I and II, the former predominate in phase III (i.e., 4:1). Moreover, there are more novel antibacterial classes among natural-product derived antibiotics compared to synthetic

Disappointment from antibacterial drug discovery in the genomic era brought a renewed interest in screening natural products (Baltz, 2008; Davies, 2011; Li & Vederas, 2009; Molinari, 2009). Chemists have been isolating and analyzing secondary metabolites from plants, fungi, and bacteria for over 200 years, yet only a small percentage of species has been addressed (Li & Vederas, 2009). Undoubtedly, the natural supply of small molecules (sometimes referred to as parvome (Davies, 2011), from the Latin *parvus* meaning small) remains vast; however, there

allow the targeted pathway to be circumvented (Gentry et al., 2003).

ones (a total of 7 new chemical scaffolds vs. 4) in the pipeline.

Oesterhelt & Sass, 2010; Projan, 2003)).

**2.3 Reappraising the natural products** 

is a problem accessing it. A majority (maybe up to 99%) of microbes, renowned for their rich and diverse metabolism, cannot be cultured in a laboratory, at least not under standard conditions (Amann et al., 1995; Li & Vederas, 2009). There are species of microbes that thrive in geographical or ecological niches, such as deep sea and thermal springs, or as symbionts of plants and animals, respectively, that still await to be explored. Besides rediscovery, a major obstacle that can impede natural product research is that some compounds are found in the environment in rather low concentrations, complicating their detection and isolation in quantities allowing structural and functional studies.

Nevertheless, the thesis that the laborious screening for natural products with antibiotic activity is still worth the effort is supported by several facts. The parvome displays structural diversity unmatched by synthetic compounds; secondary metabolites often possess numerous chiral centers and display astonishing steric complexity. Furthermore, many natural antibiotics display complex and multilayer mechanisms of action that might not have been devised by rational design. Last but not least, millions of years of evolution have optimized antibiotics with respect to affinity and specificity for their targets, as well as physicochemical properties to penetrate bacterial envelopes (Butler & Buss, 2006; Pelaez, 2006; Swinney & Anthony, 2011). Encouragingly, owing to the revival of screening for natural antimicrobials or reinspection of collections of old antibiotics in the last decade, we have witnessed attempts to develop antibiotics based on novel chemicals templates, such as lipopeptides, pleuromutilins, ramoplanins, and actinonins (Butler & Buss, 2006; Butler & Cooper, 2011). Drugs based on new scaffolds exerting novel mechanisms of action should be superior to existing antibiotic classes in the fight against multi-drug resistant pathogens (Butler & Buss, 2006). Of note, two such antibiotics have recently been approved for use in humans. Daptomycin, the first member of lipopeptide antibiotics, acts through a complex mechanism involving the disruption of the bacterial membrane leading to inhibition of DNA, RNA, and protein synthesis, and is indicated for the treatment of skin and skin structure infections caused by Gram-positive pathogens (Baltz et al., 2005). Retapamulin, a pleuromutilin type antibiotic with indications similar to those of daptomycin, selectively inhibits the P site of peptidyl transferase centre on the bacterial 50S ribosomal subunit, exhibiting a mechanism that differs from other protein synthesis-inhibiting antibiotics (Dubois & Cohen, 2010; Schlunzen et al., 2004).

It is important to realize that all small molecular weight microbial products are active even though they might not induce antibiosis at concentrations found in the environment, suggesting their role as signaling molecules (Dufour & Rao, 2011; Miao & Davies, 2010; Shank & Kolter, 2009; Wyatt et al., 2010). Remarkably, this holds true even for well established antibiotics; a number of recent studies reported specific modulation of gene expression in different bacteria when exposed to subinhibitory concentrations of various antibiotics (Davies et al., 2006; Fajardo & Martinez, 2008; Linares et al., 2006). Reevaluation of known natural products for traits other than antibiosis might thus present another route leading to antibacterial drug discovery; inhibiting the production of metabolites that provide the producing microbe with an advantage in colonizing a certain niche could prove to be a fruitful approach in designing antimicrobials (Wyatt et al., 2010).

#### **3. Where do we search for natural antibiotics?**

The search for new antibiotics compounds goes hand in hand with the discovery of new (micro)organisms producing them. For this purpose the search has continued on land and at

Future Antibiotic Agents: Turning to Nature for Inspiration 31

therapeutic value, which is why interest in screening endophytic fungi for discovery of novel metabolites, and more specifically novel antibiotics, has increased. The initial step in discovering secondary metabolites of endophytes is their successful isolation from plant materials. Then, the isolation and characterization of bioactive substances from culture filtrates is done using bioassay guided fractionation and spectroscopic methods (Strobel, 2002). For a detailed explanation on how these endophytic microorganisms are isolated the

A short selection of substances with antibiotic properties that have been found in endophytic fungi and reported so far is included in Table 1 to provide the reader with an

> Zijin Mountain, the suburb of Nanjing,

> acquisition number 237- 71-5282, Wakehurst Place,

Boraso Stream-Delta del Parana, Argentina.

Guanacaste Conservation Area in Costa Rica

Korea

China

Kangwon region,

Hainan Island, China

Jiangsu Province,

Sheyang Port on the Yellow Sea

China

UK

**plant Isolated metabolite(s)** 

colletotric acid

7,22-diene

6,22-diene

phomol

ergosta-7,22-diene 3b-hydroxy-ergosta-5-ene; 3-oxo-ergosta-4,6,8(14),22-tetraene 3b-hydroxy-5a,8a-epidioxy-ergosta-

phomopsichalasin

guanacastepenes A-O

monomethylsulochrin rhizoctonic acid guignasulfide

3β,5α,6β-trihydroxyergosta-7,22-diene

3β-hydroxy-5α,8α-epidioxy- ergosta-

periconicin A periconicin B

rhizoctonic acid monomethylsulochrin

ergosterol

ergosterol

6,22-diene

helvolic acid monomethylsulochrin

6-isoprenylindole-3-carboxylic acid 3b,5a-dihydroxy-6b-acetoxy-ergosta-

3b,5a-dihydroxy-6b-phenylacetyloxy-

reader is referred to other publications (Hallmann et al., 2006; Strobel, 2002).

idea of how many potential lead compounds there are presently at our disposal.

**strain Host plant (family) Habitat of the host** 

*Artemisia mongolica*  (Fisch. ex Bess.) Nakai

*Artemisia annua* L.

*Salix gracilistyla* var. *Melanostachys* (Salicaceae)

*Erythrina crista-galli* L.

*Daphnopsis americana*  (Thymelaeaceae)

*Hopea hainanensis* Merrill

Zucc (Taxaceae)

(Dipterocarpaceae)

*Cynodon dactylon* (L.) Pers. (Poaceae)

*Cynodon dactylon* (L.) Pers. (Poaceae)

(Fabaceae)

*Periconia* sp. OBW-15 *Taxus cuspidate* Siebold &

& Chun

(Asteraceae) ns

(Asteraceae)

**Endophytic fungal** 

*Colletotrichum gloeosporioides* (Penz.) Penz. & Sacc.

*Colletotrichum* sp.

*Phomopsis* isolate MF6031

*Phomopsis* sp. strain

*Guignardia* sp. IFB-

*Rhizoctonia* sp. strain

*Aspergillus* sp. strain

E02018

CR115

E028

Cy064

CY725

unidentified endophytic fungus

sea with great expectations. Soil microorganism exploitation has not subsided and continuous effort is put into the expanding the diversity of actinomycetes and fungi, taking advantage of little explored ecological niches and developing new ways of growing previously uncultivable strains (Harvey, 2000).

Almost all kinds of living things have the ability to produce secondary metabolites with antibiotic properties (Berdy, 2005), although this ability is not equally distributed among different species. Overall, it is clear that unicellular bacteria, eukaryotic fungi, and first of all filamentous actinomyces are the most frequent and most versatile producers. The filamentous actinomycetales species produce over 10,000 bioactive compounds, of which 7600 derived from *Streptomyces* represent the largest group (45%) of bioactive microbial metabolites. Streptomycetes are demonstrably a rich source of compounds, but no more so than other members of the actinobacteria. In 2001 Watve et al. set about to produce a mathematical model that would estimate the number of undiscovered antimicrobials from the genus *Streptomyces* (Watve et al., 2001). They found that there are still around 150,000 antimicrobials to be discovered. Theoretically speaking, this number does sound encouraging and one might expect the antibiotic pipeline to be pouring with new drugs. The reality is quite different. According to Butler and Cooper, in 2011, there were five compounds undergoing phase-III clinical trials, one compound was under NDA/MAA evaluation, 22 compounds were in phase-II, and 12 compounds in phase-I clinical trials (Butler & Cooper, 2011). Twenty of those compounds are derived from natural products. Clinical development of a drug requires certain *in vitro* activity, stability, and pharmacokinetic criteria to be met. It seems that not many of the lead secondary metabolites make it all the way to clinical trials and development and, eventually, drug approval. Nevertheless, finding an effective lead substance remains the most important starting point in antibiotic development. In the following text we give some examples on where some of these lead compounds can and have been found.
