**3.1.1 Endophytes**

Endophytes reside in tissues between living plant cells. The relationship that they establish with the plant varies from symbiotic to bordering on pathogenic. Of all of the world's plants, it seems that only a few grass species have had their complete complement of endophytes studied, although endophytic fungi have been found in each plant species examined. The estimated number of endophytic fungal species existing in nature is over one million (Petrini, 1991). As a result, the opportunity to find new and interesting endophytes among the myriad of plants is great.

Plant endophytic fungi have a special ability to produce a great number of diverse bioactive compounds, which have been implicated in protection of its host against pathogens and herbivores (Wicklow et al., 2005). These structurally diverse molecules have potential

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

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

Natural product resources, including the microbial world, are mainly unexplored both in its dimension and in the respect of geographic, ecological, and environmental points of view. There surely exist, besides the presumed numbers of microorganisms, millions of microbes living in remote and exotic parts of the world, or even the ones living in other organisms as

Endophytes reside in tissues between living plant cells. The relationship that they establish with the plant varies from symbiotic to bordering on pathogenic. Of all of the world's plants, it seems that only a few grass species have had their complete complement of endophytes studied, although endophytic fungi have been found in each plant species examined. The estimated number of endophytic fungal species existing in nature is over one million (Petrini, 1991). As a result, the opportunity to find new and interesting endophytes

Plant endophytic fungi have a special ability to produce a great number of diverse bioactive compounds, which have been implicated in protection of its host against pathogens and herbivores (Wicklow et al., 2005). These structurally diverse molecules have potential

previously uncultivable strains (Harvey, 2000).

these lead compounds can and have been found.

endophytes or symbionts that await discovery and thorough study.

**3.1 The producing organisms** 

among the myriad of plants is great.

**3.1.1 Endophytes** 

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

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


Future Antibiotic Agents: Turning to Nature for Inspiration 33

Another great source of antibiotic producers among endophytes is bacteria. Munumbicins are an example of antibacterial compounds found in these microorganisms. Gary Strobel's research group has isolated and studied the *Streptomyces* NRRL 30562 strain, which is endophytic in the medicinal plant snakevine (*Kennedia nigriscans*), native to the Northern Territory of Australia (Castillo et al., 2002). Bioassay-guided HPLC purification of the culture broth of this endophytic bacterium led to the discovery of four major components. They were characterized as four functionalized peptides named munumbicins A, B, C, and D. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had a mimimum inhibitory concentration (MIC) of 2.5 µg/ml against a methicillin-resistant strain of *Staphylococcus aureus* (MRSA), whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as *Bacillus anthracis* and multidrug-resistant *Mycobacterium tuberculosis*. The most impressive biological activity of any of the munumbicins was that of munumbicin D against the parasite *Plasmodium falciparum*. However, in 2006, they reported that some of the munumbicins are identical to the better known antibiotics, the actinomycins (Castillo et al. 2006). Further effort resulted in the isolation of several novel antibiotics from *Streptomyces* NRRL 30562 with wide-spectrum biological activity that were termed munumbicin E-4 and E-5 (Castillo et al. 2006). Both compounds were tested alongside vancomycin against *Escherichia coli* and MRSA. The MIC of munumbicin E-5 against *E. coli* was 16 µg/ml, while the MIC for vancomycin was

128 µg/ml. The MICs were 16 and 2 µg/ml against MRSA, respectively.

Cragg, 2004).

**3.1.2 Insects** 

Other antibiotic compounds of different chemical structures such as the bafilomycins (Yu et al., 2011), kakadumycins (Castillo et al., 2003) and many others are also produced by endophytic *Streptomyces* strains, making these bacteria worth investigating. The ability to make bioactive small molecules is not exclusive to microbes. Plants are rich sources of a great variety of compounds, but the original producer of those might be questionable. Opinions on this subject are divided and nobody can say for certain how many microbial metabolites/antibiotics considered today as marine animal or plant products, are produced, in fact, by symbiotic microbes in marine invertebrates and by endophytic fungi or bacteria living in the vascular plants. It has become generally accepted that at least for some compounds isolated from marine invertebrates, and in several cases from higher plants, the actual producers are the symbiotic microbes; bacteria, cyanobacteria, algae, or endophytic fungi. Indeed, several bioactive metabolites (e.g. taxol, bryostatin, theopalauamide, caphalomannin, etc.) have been proven to originate from symbiotic or endophytic microbes and not the "higher" (host) organisms (Berdy, 2005; Newman &

Insects represent 80% of all fauna and are the most widespread group within the animal kingdom. Furthermore, some of these organisms such as cockroaches live in the filthiest

An investigation of the potential antibacterial activity in various tissues of the desert locust (*Schistocerca gregaria*) and American cockroach (*Periplaneta americana*) was undertaken at the School of Veterinary Medicine and Science, University of Nottingham. Brain lysates of locust and cockroach exhibited powerful broad-spectrum antibiotic

places known to man and thrive in such conditions (Lee et al., 2011).


Table 1. Plant endophytic fungi producing metabolites with antibacterial activity. ns – not specified.

Jiangsu and Shandong provinces,

Harwan, Jammu and Kashmir, India

Sichuan University, Chengdu, Sichuan Province, China

F.W. Schmidt (Asteraceae) Alexandria, Egipt 3-O-methylalaternin

central highlands of Papua New Guinea

southern hillside of the Zijin Mountain in the eastern suburb of Nanjing, China

Yunnan Province,

coastal area of the Baltic Sea, Ahrenshoop, Germany

Playa del Ingles, Gomera, Spain

Dong Zhai Gang Mangrove Garden on Hainan Island, China

Varanasi district,

India javanicin

China

China

**plant Isolated metabolite(s)** 

hypericin emodin

broth

chlorogenic acid

altersolanol A

phomodione usnic acid cercosporamide

cerebroside 1 cerebroside 2

koninginin A

ribonucleoside

fusidienol A

xanalteric acid I xanalteric acid II altenusin

hydroxyphthalide

microsphaeropsone A microsphaeropsone C citreorosein

enoate shikimic acid cytosine ribonucleoside

7-amino-4-methylcoumarin

crude ethanol extract of fermentation

(E)-2,3-dihydroxypropyl octadec-9-

a compound considered to be adenine

enone (oxidized microsphaeropsone A)

8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester

4-hydroxyphthalide; 5-methoxy-7-

(3R,4R)-cis-4-hydroxymellein

Kunming, China helvolic acid

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

*Paris polyphylla* var. *yunnanensis* (Franch) Hand.-Mazz (Trilliaceae)

*Hypericum perforatum* L. (Hypericaceae)

*Eucommia ulmoides* Oliver (Eucommiaceae)

*Saurauia scaberrinae* (Actinidiaceae )

(Fagaceae)

*Quercus variabilis* Blume

*Panax notoginseng* (Burkill ) F.H.Chen ex C.Y.Wu & K.M.Feng (Araliaceae)

*Melilotus dentatus* (Waldst. & Kit.) Pers. (Fabaceae)

*Lycium intricatum* Boiss.

*Zygophyllum fortanesii*

*Sonneratia alba* J.E. Smith (Sonneratiaceae)

*Azadirachta indica* A. Juss.

(Meliaceae)

(Zygophyllaceae) Gomera, Spain

<sup>7092</sup>*Erica arborea* L. (Ericaceae) Gomera, Spain 3,4-dihydroglobosuxanthone A

Table 1. Plant endophytic fungi producing metabolites with antibacterial activity.

(Solanaceae)

*Ginko biloba* L. (Ginkgoaceae)

*Ampelomyces* sp. *Urospermum picroides* (L.)

**Endophytic fungal** 

*Pichia guilliermondii* 

*Thielavia subthermophila*  INFU/Hp/KF/34B

unidentified endophytic fungal strains

Ppf9

*Xylaria* sp. YX-28

Twenty-nine

*Phoma* sp. NG-25

*Fusarium* sp. IFB-121

PRE-5

Unidentified

*Trichoderma ovalisporum*

Ascomycete endophytic fungus strain 6650

*Microsphaeropsis* sp. strain 8875

*Microsphaeropsis* sp. strain 7177

*Microdiplodia* sp. strain

*Alternaria* sp. strain

*Chloridium* sp. (J.F.H. Beyma) W. Gams & Holubova-Jchova

ns – not specified.

JCM9.2

Another great source of antibiotic producers among endophytes is bacteria. Munumbicins are an example of antibacterial compounds found in these microorganisms. Gary Strobel's research group has isolated and studied the *Streptomyces* NRRL 30562 strain, which is endophytic in the medicinal plant snakevine (*Kennedia nigriscans*), native to the Northern Territory of Australia (Castillo et al., 2002). Bioassay-guided HPLC purification of the culture broth of this endophytic bacterium led to the discovery of four major components. They were characterized as four functionalized peptides named munumbicins A, B, C, and D. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had a mimimum inhibitory concentration (MIC) of 2.5 µg/ml against a methicillin-resistant strain of *Staphylococcus aureus* (MRSA), whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as *Bacillus anthracis* and multidrug-resistant *Mycobacterium tuberculosis*. The most impressive biological activity of any of the munumbicins was that of munumbicin D against the parasite *Plasmodium falciparum*. However, in 2006, they reported that some of the munumbicins are identical to the better known antibiotics, the actinomycins (Castillo et al. 2006). Further effort resulted in the isolation of several novel antibiotics from *Streptomyces* NRRL 30562 with wide-spectrum biological activity that were termed munumbicin E-4 and E-5 (Castillo et al. 2006). Both compounds were tested alongside vancomycin against *Escherichia coli* and MRSA. The MIC of munumbicin E-5 against *E. coli* was 16 µg/ml, while the MIC for vancomycin was 128 µg/ml. The MICs were 16 and 2 µg/ml against MRSA, respectively.

Other antibiotic compounds of different chemical structures such as the bafilomycins (Yu et al., 2011), kakadumycins (Castillo et al., 2003) and many others are also produced by endophytic *Streptomyces* strains, making these bacteria worth investigating. The ability to make bioactive small molecules is not exclusive to microbes. Plants are rich sources of a great variety of compounds, but the original producer of those might be questionable. Opinions on this subject are divided and nobody can say for certain how many microbial metabolites/antibiotics considered today as marine animal or plant products, are produced, in fact, by symbiotic microbes in marine invertebrates and by endophytic fungi or bacteria living in the vascular plants. It has become generally accepted that at least for some compounds isolated from marine invertebrates, and in several cases from higher plants, the actual producers are the symbiotic microbes; bacteria, cyanobacteria, algae, or endophytic fungi. Indeed, several bioactive metabolites (e.g. taxol, bryostatin, theopalauamide, caphalomannin, etc.) have been proven to originate from symbiotic or endophytic microbes and not the "higher" (host) organisms (Berdy, 2005; Newman & Cragg, 2004).

#### **3.1.2 Insects**

Insects represent 80% of all fauna and are the most widespread group within the animal kingdom. Furthermore, some of these organisms such as cockroaches live in the filthiest places known to man and thrive in such conditions (Lee et al., 2011).

An investigation of the potential antibacterial activity in various tissues of the desert locust (*Schistocerca gregaria*) and American cockroach (*Periplaneta americana*) was undertaken at the School of Veterinary Medicine and Science, University of Nottingham. Brain lysates of locust and cockroach exhibited powerful broad-spectrum antibiotic

Future Antibiotic Agents: Turning to Nature for Inspiration 35

Fig. 2. Structures of pyrrhocoricin, pestalone, psammaplin A, and pleuromutilin.

use for drug discovery in the future (Cueto et al., 2001).

Research conducted with a marine fungus of the genus *Pestalotia* isolated from the surface of the brown alga *Rosenvingea* sp. collected in the Bahamas, lead to the discovery of pestalone (Fig. 2), a new chlorinated benzophenone antibiotic, which has potent antibiotic activity against MRSA, with a MIC of 37 ng/mL, and vancomycin-resistant *Enterococcus faecium*, with a MIC of 78 ng/mL (Cueto et al., 2001). The potency of this agent toward drug-resistant pathogens suggests that pestalone should be assessed in more advanced infectious diseases animal models. Interestingly, pestalone is produced in the mixed fermentation of a marine fungus, *Pestalotia* sp. (strain CNL-365) and an unidentified, antibiotic-resistant marine bacterium (CNJ-328), highlighting the complex dependence of metabolite biosynthesis on culture conditions and the potential for enhanced antibiotic production through crossspecies induction. This observation clearly demonstrates that pestalone is a product of fungal biosynthesis in response to an external trigger, suggesting that this method may have

properties (>90% bactericidal effects) against MRSA and neuropathogenic *E. coli* K1, strain E44 (a cerebrospinal fluid isolate from a meningitis patient, O18:K1:H7), a spontaneous rifampicin-resistant mutant (Lee et al., 2011). A preliminary test suggested that the active substance is proteinaceous in nature. Brain lysates had no cytotoxic effects on human brain microvascular endothelial cells, suggesting that the putative target(s) is not present in eukaryotic cells. By combining size-exclusion spin columns and fast-performance liquid chromatography, eight different molecules (3–10 kDa in molecular mass) in brain lysates were identified that were toxic both to MRSA and neuropathogenic *E. coli* K1.

Higher insects protect themselves against bacterial infection by rapid synthesis of a battery of potent antibacterial peptides. Antimicrobial peptides (AMPs) have become recognized as important components of the nonspecific host defense or innate immune system in a variety of organisms including bacteria, fungi, plants, insects, birds, crustaceans, amphibians, and mammals (Zasloff, 2002). In order to overcome the problem of multi-resistant pathogenic bacteria, it is imperative to discover and clinically develop agents selectively toxic to bacteria that act on new targets which have not yet experienced selective pressure in the clinical setting. The research group of Laszlo Otvos has prepared derivatives of native proline-rich antibacterial peptides which exhibit these required features. As a lead they used pyrrhocoricin (Fig. 2), a peptide their group originally isolated from the European sapsucking bug *Pyrrhocoris apterus* (Cociancich et al., 1994). In a couple of publications they report that pyrrhocloricin is non-toxic to eukaryotic cells and healthy mice, has good activity against model bacterial strains *in vitro* and when administered intravenously *in vivo*, and can protect mice from systemic *E. coli* challenge (Cudic et al., 2002; Cudic et al., 2003; Otvos et al., 2000a). Although pyrrhocoricin is toxic to infected animals at a high dose (50 mg/kg), its derivative in which the peptide is protected from exopeptidase cleavage by replacement of the N-terminal Val1 with 1-amino-cyclohexane-carboxylic acid and the C-terminal Asn20 with acetylated 2,3-diamino-propionic acid lacks this high dose toxicity and shows improved protease resistance, while maintaining the *in vitro* and *in vivo* efficacy over a broad concentration and dose range. Even more importantly, pyrrhocoricin and the derivative seem to have a completely new mechanism of action which makes the likelihood of fast accumulation of resistant strains insignificant. Native pyrrhocoricin kills the sensitive species by binding to DnaK, the 70-kDa bacterial heat shock protein (Otvos et al., 2000b). Remarkably, pyrrhocoricin does not bind to the human equivalent protein Hsp70, indicating the potential of this peptide as a drug lead to treat human or animal infections.

A list of antimicrobial peptides in clinical trials was published in 2004 (Andres & Dimarcq, 2004), and to date none of the peptides described has obtained FDA approval for any of the various clinical indications.

#### **3.1.3 Marine (micro)organisms**

The annual Marine natural products report has been published continuously since 1984 (until 2002 by John Faulkner (Faulkner, 2002) and later by Blunt et al. (Blunt et al., 2011)). The 2011 report states that 1011 new compounds of marine origin were described in literature in 2009 alone, proving that the oceans are a vast resource of diverse natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria (Donia & Hamann, 2003).

properties (>90% bactericidal effects) against MRSA and neuropathogenic *E. coli* K1, strain E44 (a cerebrospinal fluid isolate from a meningitis patient, O18:K1:H7), a spontaneous rifampicin-resistant mutant (Lee et al., 2011). A preliminary test suggested that the active substance is proteinaceous in nature. Brain lysates had no cytotoxic effects on human brain microvascular endothelial cells, suggesting that the putative target(s) is not present in eukaryotic cells. By combining size-exclusion spin columns and fast-performance liquid chromatography, eight different molecules (3–10 kDa in molecular mass) in brain lysates

Higher insects protect themselves against bacterial infection by rapid synthesis of a battery of potent antibacterial peptides. Antimicrobial peptides (AMPs) have become recognized as important components of the nonspecific host defense or innate immune system in a variety of organisms including bacteria, fungi, plants, insects, birds, crustaceans, amphibians, and mammals (Zasloff, 2002). In order to overcome the problem of multi-resistant pathogenic bacteria, it is imperative to discover and clinically develop agents selectively toxic to bacteria that act on new targets which have not yet experienced selective pressure in the clinical setting. The research group of Laszlo Otvos has prepared derivatives of native proline-rich antibacterial peptides which exhibit these required features. As a lead they used pyrrhocoricin (Fig. 2), a peptide their group originally isolated from the European sapsucking bug *Pyrrhocoris apterus* (Cociancich et al., 1994). In a couple of publications they report that pyrrhocloricin is non-toxic to eukaryotic cells and healthy mice, has good activity against model bacterial strains *in vitro* and when administered intravenously *in vivo*, and can protect mice from systemic *E. coli* challenge (Cudic et al., 2002; Cudic et al., 2003; Otvos et al., 2000a). Although pyrrhocoricin is toxic to infected animals at a high dose (50 mg/kg), its derivative in which the peptide is protected from exopeptidase cleavage by replacement of the N-terminal Val1 with 1-amino-cyclohexane-carboxylic acid and the C-terminal Asn20 with acetylated 2,3-diamino-propionic acid lacks this high dose toxicity and shows improved protease resistance, while maintaining the *in vitro* and *in vivo* efficacy over a broad concentration and dose range. Even more importantly, pyrrhocoricin and the derivative seem to have a completely new mechanism of action which makes the likelihood of fast accumulation of resistant strains insignificant. Native pyrrhocoricin kills the sensitive species by binding to DnaK, the 70-kDa bacterial heat shock protein (Otvos et al., 2000b). Remarkably, pyrrhocoricin does not bind to the human equivalent protein Hsp70, indicating

were identified that were toxic both to MRSA and neuropathogenic *E. coli* K1.

the potential of this peptide as a drug lead to treat human or animal infections.

and from marine bacteria and cyanobacteria (Donia & Hamann, 2003).

various clinical indications.

**3.1.3 Marine (micro)organisms** 

A list of antimicrobial peptides in clinical trials was published in 2004 (Andres & Dimarcq, 2004), and to date none of the peptides described has obtained FDA approval for any of the

The annual Marine natural products report has been published continuously since 1984 (until 2002 by John Faulkner (Faulkner, 2002) and later by Blunt et al. (Blunt et al., 2011)). The 2011 report states that 1011 new compounds of marine origin were described in literature in 2009 alone, proving that the oceans are a vast resource of diverse natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs,

Fig. 2. Structures of pyrrhocoricin, pestalone, psammaplin A, and pleuromutilin.

Research conducted with a marine fungus of the genus *Pestalotia* isolated from the surface of the brown alga *Rosenvingea* sp. collected in the Bahamas, lead to the discovery of pestalone (Fig. 2), a new chlorinated benzophenone antibiotic, which has potent antibiotic activity against MRSA, with a MIC of 37 ng/mL, and vancomycin-resistant *Enterococcus faecium*, with a MIC of 78 ng/mL (Cueto et al., 2001). The potency of this agent toward drug-resistant pathogens suggests that pestalone should be assessed in more advanced infectious diseases animal models. Interestingly, pestalone is produced in the mixed fermentation of a marine fungus, *Pestalotia* sp. (strain CNL-365) and an unidentified, antibiotic-resistant marine bacterium (CNJ-328), highlighting the complex dependence of metabolite biosynthesis on culture conditions and the potential for enhanced antibiotic production through crossspecies induction. This observation clearly demonstrates that pestalone is a product of fungal biosynthesis in response to an external trigger, suggesting that this method may have use for drug discovery in the future (Cueto et al., 2001).

Future Antibiotic Agents: Turning to Nature for Inspiration 37

antibiotic, while BC-3781 successfully completed a phase-II clinical trial for the treatment of acute bacterial skin and skin structure infections (ABSSSI) (US National Institutes of Health, 2011). Nabriva's lead product BC-3781 is the first of a new class of systemically available pleuromutilin antibiotics for the treatment of serious skin infections and pneumonia. BC-

Although the number of antibiotics present in nature may truly be huge, many of them are already known or will not be usable (i.e., will not display selective toxicity to bacteria, will be too weak, or will lack the desired pharmacokinetic properties) (Pelaez, 2006). Yet historically, the development of antibiotics from natural templates has seen an unprecedented gain compared to the *de novo* synthesis. The conventional discovery process of antibiotics from the pool of microbial natural products requires having a given microorganism grown in conditions appropriate to induce the production of (the desired) metabolite, which is then extracted and tested in a screen able to detect it as a hit. Finally,

Identification of novel antibiotic types that occur in relatively low frequency in nature clearly requires innovative detection and characterization techniques. Numerous promising microbiological approaches supplemented with bioinformatic, genetic, and structural methods have been developed over the last decade to address the issue (Fig. 3).

Fig. 3. Postgenomic approaches in antibiotic discovery (adapted from (Davies, 2011)).

3781 is being developed for both oral and intravenous formulations.

the compound has to be isolated from the original mixture and identified.

**4. How do we search for natural antibiotics?** 

Sponges alone produce more than 3300 antibiotics and other bioactive compounds. It is noteworthy to mention that these isolated "animal" compounds very frequently show surprising analogy to microbial or algal products. As with the secondary metabolites produced by plants and their endophytes, it is not surprising that in numerous occasions the active compounds isolated from sponges proved to be derived from the microorganisms living in symbiosis with their host (Berdy, 2005). Marine microbes are particularly attractive because of the high potency required for bioactive compounds to be effective in the marine environment, due to the diluting effect of seawater (Zhang et al., 2005).

Psammaplin A (Fig. 2) is a symmetrical bromotyrosine-derived disulfide natural product isolated from the *Psammaplysilla* sponge (Arabshahi & Schmitz, 1987), with *in vitro* antibacterial activity against MRSA. Based on the structure of psammaplins, Nicolaou et al. produced a library of 3,828 compounds. Six of these optimized antibacterial agents possessed more than 50-fold higher activities than the natural product, demonstrating MIC levels in methicillin-resistant/intermediate vancomycin-resistant strains of *S. aureus* at less than 1µg/ml. In order to construct these heterodimeric disulfide analogues they used a novel combinatorial disulfide exchange strategy, thus demonstrating the power of modern combinatorial techniques when applied to a base active structure from nature (Newman & Cragg, 2004; Nicolaou et al., 2001a; Nicolaou et al., 2001b). Most significantly, a number of these agents exhibited increased selectivity against bacterial cells over fibroblasts and lymphocytes as compared to the natural product.

In similar efforts of the marine natural products community, many antibacterial agents have been identified from sponges (Laport et al., 2009). Despite their high number, none of them has yet been involved in clinical trial as an antibacterial agent.
