**4. Challenges**

**3.3. Microorganisms**

Microorganisms were identified early on as sources of valuable natural products as evidenced by the discovery of penicillin by from the fungus *Penicillium rubens* by Alexander Fleming in 1928 [100]. Historically, microorganisms (amongst them mostly bacteria and fungi) have played an important role in providing new structures, like antibiotics for drug discovery and development. The terrestrial microbial populations are immensely diverse which is also reflected in the number of compounds and metabolites isolated from these microorganisms. As mentioned above, the similarity of many compounds from marine invertebrates like sponges, ascidians, soft corals and bryozoans to those isolated from terrestrial microbes led to the hypothesis that associated microorganisms might be responsible for their production. Over time it became more and more evident, that a significant number of marine natural products are actually not produced by the originally assumed invertebrate but rather by microbes living in symbioses with their invertebrate host [92, 101]. In some instances it could indeed be demonstrated early on that the isolated marine microbes are the original source of the new compounds or secondary metabolites discovered and in recent years marine bacteria have emerged more and more as a source of NCEs [58, 91, 94, 95]. Besides bacteria, marine fungi and deep-sea hydrothermal vent microorganisms are reported to produce bioactive com‐ pounds and metabolites [91, 94]. Deep-sea vent sites offer harsh conditions in depth below 200 meters with complete absence of light, pressures in excess of 20 atmospheres, temperatures of up to 400°Celius, pH extremes and high sulfide concentrations and are populated by highly

Unique microorganisms are abundant on land, in freshwater and all areas of the ocean. However, the enormous biological diversity of free-living and symbiotic marine microbes has so far only been explored to a very limited extent. The estimates extrapolate the number of marine species to at least a million, but for marine microbial species, including fungi and bacteria, the estimated numbers reach as high as tens or even hundreds of millions [23]. Over 74,000 known species of fungi are reported including around 3,000 aquatic species of which only about 465 are described as marine species, but a vast geographical area has not yet been sampled and estimates for the potential total number of species reach from 0.5 to 9.9 million with about 1.5 million considered as most realistic [103, 104]. The overlap is assumed to be relatively high between species in terrestrial and freshwater habitats, but not between these two and the marine habitat [104]. Nevertheless, a large percentage of the over 270 secondary metabolites isolated from marine fungi resembles analogues of compounds previously discovered from terrestrial fungi but some of the new substances identified exhibited potent activities against tumor cells, microbes or bacteria like methicillin-resistant *Staphylococcus aureus* (MRSA) or even antifouling properties [59, 105]. In comparison to bacteria, fungi appear to be rare in marine environments and few marine fungi isolates exist in culture [106]. Marine bacteria are assumed to constitute approximately 10% of the living biomass carbon and inhabit mainly sediments but can also be found in open oceans and in association with marine organisms [90, 91, 94]. Many marine invertebrates are associated with large amounts of epibiotic and endobiotic microorganisms and for many sponges bacteria can make up to 40% of the animal biomass and even resemble new species [97]. In fact it is assumed that almost all

dense and unique, biologically diverse communities [91, 94, 102].

16 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Natural products, although a valuable and precious resource, also come with their fair share of challenges in a variety of aspects. As mentioned before, one of the major issues concerning the use of natural products are the difficulties associated with obtaining sufficient amounts of material pure enough for discovery and development activities. If a compound is derived from a plant growing only in small quantities or remote locations or a marine organism residing in great depth or difficult to access regions, re-supply becomes a problem.

The threat of losing potentially valuable natural sources of pharmacologically active ingredi‐ ents is constantly increasing due to the threat of extinction by deforestation of large landmass‐ es and environmental pollution in remote areas as well as global warming [85]. It is estimated that about 70% of the supply of herbal raw material for Ayurveda and other homeopathic medicines inIndiacomes fromthewild[82,85].Tomeetthe increasingdemandforrawmaterial, toconservewildresources,andtoreducethepotentialvariabilityintheactiveingredientcontent in medicinal plants from different collection areas, it is important to implement more control‐ led cultivation programs to ensure quality and to protect resources [82, 85].

Tissues of marine invertebrates present unique problems for extraction, because of their high water and salt content, and the promising compounds may be present only in low amounts and/or can be very difficult to isolate. Sponges and their microbial fauna are mostly not suitable for culture, and the compounds of interest need to be extracted and purified from specimens collected in the wild [17, 93]. Marine organisms and microbes constitute a valuable potential source of NCEs and structural templates for drug discovery in the future, but may necessitate tons of raw material to isolate milligram to gram amounts of the compound of interest [97]. This difficulty combined with the challenges for synthetic approaches to obtain significant quantities of potential new drug candidates, based on their often highly complex structures, are obstacles that can hamper their use in discovery and development [58, 97]. In some cases, supply issues could be resolved by semi-synthesis or total synthesis of the compound, the development of synthetic analogs with more manageable properties, or by design of a pharmacophore of reduced complexity, which can then be synthesized [17, 92, 97]. Fragments or synthetic analogs with simplified structures may retain bioactivity or even show improved activity towards the target [12].

Furthermore, environmental aspects can constitute significant hurdles for supplying material for discovery and development as the product may stem from an endangered species or the wild collection of the producing species may be detrimental to its originating terrestrial or marine ecosystem. Additionally, as mentioned above, because of their low abundance many compounds of interest from natural sources need to be extracted and purified from large quantities of specimen collected in the wild, which in turn carries the risks of over-exploitation and habitat destruction [17, 93]. Radjasa *et al.* provide a positive outlook for marine ecosystems and state "*There is optimism for the future because the international marine bioorganic community clearly recognizes that invertebrates must be harvested and studied in an environmentally sustainable manner"* [92]. Although aquaculture of marine species or culture of bacteria seem like logical alternative sources to obtain product, they are not viable avenues in most cases because it proves difficult to impossible to culture the source organisms (especially invertebrates and/or their microbial symbionts like bacteria etc.) or they may not produce the compound of interest under the given culture conditions [12, 17, 95, 97]. Findings indicate, that the bacterial composition on invertebrates is largely independent from sponge taxonomy or locality of collection and the bacteria most likely are contaminants from the ocean water rather than specific symbionts, which further exacerbates the cultivation problem [97, 108].

Another challenge can result from redundant activity determination in assay systems and the mixed composition of natural product extracts. With over 150,000 known small molecules characterized from natural sources, previously known natural products are often re-isolated in the course of bioassay-guided fractionation [84]. While this may be acceptable if the biological activity is new, it is frustrating to waste resources on the *de novo* structure elucidation of known compounds. Furthermore, not all compounds contained in natural product extracts are drug leads and it is extremely desirable to remove "nuisance compounds" like tannins, phorbol esters, saponins, and anionic polysaccharides; the latter, for instance, being highly active in cellular HIV bioassays [35].

Last, but not least, intellectual property rights can pose a significant hurdle that is difficult to manage. In general, patent protection can be obtained if the active principles derived from natural sources have novel structures and relevant biological activity. However, as mentioned before, additional handicaps may arise in the context of developing and marketing products from natural sources in the form of potentially significant intellectual property issues as well as, possibly, very difficult and costly negotiations to obtain agreements to collect and develop natural products from species collected in foreign countries [25, 26, 35].
