Australian biodiversity via its plants and marine organisms. A high-throughput screening approach to drug discovery.

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High-throughput screening (HTS) of extracts of Australian plants and marine organisms commenced in our laboratory in 1994. The biota collections commenced in late 1993. The collection has in excess of 30 000 biota samples including over 16 000 biota
  Pure Appl.Chem. , Vol.74, No.4, pp.519–526, 2002.DRAFT ©2002 IUPAC519 Australian biodiversity via its plants and marineorganisms.A high-throughput screeningapproach to drug discovery* Ronald J.Quinn 1,‡ , Priscila de Almeida Leone 1 ,Gordon Guymer 2 ,and John N.A.Hooper 3 1 AstraZeneca R&D Griffith University, Brisbane, Queensland, 4111, Australia;  2  Queensland Herbarium, Brisbane, Queensland, 4066, Australia;  3  Queensland Museum, Brisbane, Queensland, 4101, Australia   Abstract  : High-throughput screening (HTS) of extracts of Australian plants and marineorganisms commenced in our laboratory in 1994. The biota collections commenced in late1993. The collection has in excess of 30 000 biota samples including over 16 000 biota sam-ples of vascular plants, algae, and macro fungi from Queensland, and over 4000 marine inver-tebrates from Australian waters. The plant collection represents ~9 % of the world speciesdiversity of higher plants, with representation from 73 % of the world’s plant families. Themarine collection contains ~10 % of the world diversity of sponges, ~10 % of the worlddiversity of ascidians, and ~5 % of the world diversity of soft corals and gorgonians.The lecture will highlight some of the advances to knowledge about Australian biodi-versity as a result of the HTS project, discuss drug discovery using HTS, and give someexamples of the chemistry arising from the screening of the extracts.Australia is one of 12 megadiverse countries (Mexico, Colombia, Ecuador, Peru, Brazil, Zaire,Madagascar, China, India, Malaysia, Indonesia, Australia). These countries have greater than 70 % of world biodiversity [1]. Australia is a party to the Convention on Biological Diversity, which states thatparticipating countries have sovereign rights over their own biological resources. Historically, natural products have been a significant contributor to drug discovery. In 1996, eightof the 20 top-selling pharmaceuticals were natural product-derived. In the period 1983–1994, 203 outof the 520 NCEs introduced worldwide were derived from natural sources [2]. There has been a highsuccess rate of NPS in oncology with 61 % of NCEs introduced between 1983–1994 being natural prod-uct-derived [2]. Taxol/taxotere (from plants) has achieved combined No. 1 sales in oncology. Diversityanalysis by Bayer of its 350 000-compound synthetic library plus other databases indicated 40 % of nat-ural products were not represented by the investigated synthetics [3]. Complementarity of diversity isobtained by a combined compound and natural product library.It is estimated that the diversity of living organisms on Earth is between 5 and 100 million species(only 1.4 million have actually been described). Bacteria and arthropods make up the vast majority of this biodiversity (~90 %). It has also been estimated that 90–99 % of bacteria cannot be cultured usingtechniques available today. Natural product drug discovery programs have traditionally targeted thoseorganisms that can be easily collected or cultured, and include plants, dominant (>1 kg) marine organ-isms, and culturable microbes. The AstraZeneca–Griffith University (AZGU) collection asset com- * Lecture presented at 3 rd IUPAC International Conference on Biodiversity (ICOB-3), Antalya, Turkey, 3–8 November 2001. ‡ Corresponding author  prises 35 220 biota and includes collections of vascular plants, algae, and macro fungi (24 017) fromQueensland (18 732), Southern China (6314), and Papua New Guinea (2501), marine invertebrates fromAustralian and Antarctic waters (5871), soil and aquatic fungal extracts from India (1802). The plant collection represents ~9 % of the world species diversity of higher plants, with repre-sentation from 73 % of the world’s plant families. The marine collection contains ~10 % of the worlddiversity of sponges, ~10 % of the world diversity of ascidians, and ~5 % of the world diversity of softcorals and gorgoniansThe natural product drug discovery program in our laboratory consists of plant and marine col-lection, extraction, high throughput screening of the extracts, identification of active extracts, bioassay-directed fractionation, isolation of the active constituents, structure elucidation, and lead optimizationusing medicinal chemistry. At the commencement of the project we envisaged the need to isolate only1 mg of material for structure elucidation and biological evaluation in the screen of interest.The requirement for a small amount of isolated compound established the logistics of our naturalproduct drug discovery project. Small-scale extraction and chromatography were integral to theapproach. The gains in efficiency with miniaturization have been substantial, allowing the isolation of over 600 bioactive compounds.In order to be successful in any screening operation, the vast amount of data generated by HTSof extracts, fractions, or compounds must be harnessed. We have opted to write our own software. Thisdecision was based on evaluation of commercial packages and a decision that in-house developmentwould allow as good or better customization in the initial stages but, critically, would allow easier adap-tation to future unforeseen requirements. The software package, HiTbaSe, has served us extremely well.HiTbaSe is a client/server-based system with graphical client software for data entry and query. Allinformation is stored in SQL tables on an Oracle server. Assays, experiments, and plate templates canbe set up via an intuitive user interface. With the aid of a user-configurable import system HiTbaSereads raw screening data from any file format. HiTbaSe provides a comprehensive quality control anddecision-making environment via its “Activity Centre”. Hit Selection is performed by “Active Search”,a powerful visualization and filtering interface. The system also tracks sample status and status history,providing powerful list management options. The Plate Wizard provides a logical way of submittinglow- to high-volume follow-up experiments. Performance is high due to optimized ADO connectivitydirectly with Oracle. A performance test, of rank ordering a 140 000 well screen in descending ordertook 35 s—on a 400-MHz Sun Enterprise 250 Oracle server.HTS has focused on receptors, enzymes, and mechanism-based cellular assays in the area of car-diovascular, respiratory, inflammation, gastrointestinal, pain control, CNS, infection, and oncology.HTS commenced in July 1994. Continuous screening, where a panel of around 20 screens wasassayed each year against 10 000 extracts was implemented from 1994–1997. The process identifiedpotential lead compounds, however this required a screen lifetime of two years to evaluate 20 000extracts. This was not in pace with the need for rapid identification of leads. The most interesting orpotent extract may not be identified until the later part of the screening.In 1997, with the extract collection at 30 000 extracts, continuous screening was replaced by cam-paign screening. Campaign screening allowed the entire extract library to be screened in a semi-auto-mated manner within four weeks. Between 1997 and 2000, the extract library increased greater thanthreefold while the time to assay the extract library either remained at the 1997 level or in most caseswas able to be decreased. This was accomplished by the judicious purchase of state-of-the-art roboticliquid handling and assay building workstations that allow 100 384-well plates per day (depending onscreen technology). Four robotic workstations (assay building stations) are in place.The first 100 000 extract campaign was achieved in May 2000. In 2000, the number of wells(assay points) achieved in primary screening was 273 % of the number in 1999. Also in 2000, the num-ber of extracts per screen increased substantially and was 244 % of the 1999 figure. In terms of biodiversity conservation, it is valuable to examine the effect of this miniaturization.A comparison of biodiversity discovery programs of the 1970s with current efforts also highlights the R.J.QUINN et al.  ©2002 IUPAC, Pure and Applied Chemistry  74, 519–526520  increased biodiversity conservation value of today’s research. The Roche Research Institute of MarinePharmacology (RRIMP) operated in Sydney between 1974 and 1981. Sample collections were10 kg of wet sponge. This amount was a function of the screening, isolation, and structure elucidation require-ments. Screening was based on animal models and isolated tissue preparations. Six extracts srcinatingfrom two species were screened each week against a panel of screens. In total, around 2100 extractswere screened over seven years against a panel of screens. The AZGU program collects around 100 gof biota. Extraction of 200 mg of biota provides sufficient extract for screening against receptors,enzymes, and mechanism-based cellular targets. This has allowed the screening of 100 000 extractsagainst the targets. In 96-well format, screening the extract from 100–1000 µ g of sample per well (usually 200 µ L)allows 200 mg of sample to give 2000–200 assays points. The more recent introduction of 384-well for-mat allowed a reduction of assay volume to around 50 µ L, so that 200 mg of biota at 25–250 µ g perwell provides 8000–800 assay points. New knowledge of the marine fauna The sea contains a much greater potential source of genetic diversity than does the land, with a muchlarger variety of life forms (body plans, or phyla) that live there. Of the 28 marine phyla about 33 000species have been described from the Australian EEZ (excluding vertebrates apart from fishes) (ABRS,2001). This fauna currently known to science is estimated to represent less than one-third of the totalnumber of species actually living in Australian waters, which in turn is also estimated to comprise about30 % of the world’s marine fauna. The Australian marine biota is also highly unique, with many species not found anywhere else inthe world. It has both Tethyan and Gondwanan srcins, with the former responsible for the mainly trop-ical species that are supposedly widely distributed throughout the Indo-west Pacific (an equivocal claimthat still remains largely untested), and the latter for the more highly endemic, mainly southern faunas.Moreover, in the transition zones, where these northern and southern faunas overlap, there has been asubstantial mixing of species that has produced many endemic regional marine faunas, on both the eastand west coasts of the continent. Similarly, there are over 60 marine bioregions in the Australian EEZ[4], each characterized by particular suites of biota and physical-chemical features that set them apartfrom other regions, together representing a huge diversity of ecosystems. Consequently, as for the ter-restrial fauna and flora, the Australian marine biota is also megadiverse. However, despite these claims,we still know very little about our marine biota, with about 40 % of marine families lacking even basicdata. Over the past decade we have come a lot closer to understanding the magnitude and diversity of some of these marine phyla, thanks largely to biodiscovery/bioprospecting for new chemical com-pounds. Since the AZGU “biodiscovery” collection programs commenced in 1993 our knowledge of the biodiversity of sessile marine invertebrates (animals fixed to the seabed) has increased phenome-nally, especially of the lower phyla containing sponges, soft corals, gorgonians, and bryozoans, as wellas members of the higher invertebrates, viz., tunicates, or ascidians. These taxa are among the mostinteresting and bioactive targets for pharmaceutical research worldwide [5]. During the AZGU pro-gram over 8000 marine specimens have been collected, of which 5858 specimens (weighing more than100 g wet wt) were used for screening operations. These collections consist of about 3500 species and2005 species, respectively, collected from over 1000 individual sites throughout tropical Australia. Theproportion of specimens (and species) for the target phyla is as follows: •Phylum Porifera. Collections of sponges currently consist of 4420 specimens >100 g wet wt, rep-resenting 1247 species. Additional collections of ~2200 specimens <40 g wet wt have also beenmade during this program, representing ~1500 species, mainly the small encrusting and crypticspecies that were too small to produce adequate chemical extracts for screening operations.  ©2002 IUPAC, Pure and Applied Chemistry  74, 519–526 Australian biodiversity via its plants and marine organisms  521  •Phylum Cnidaria. These collections consist of 681 specimens of soft corals, gorgonians, jellyfish,anemones, and related taxa, consisting of 292 species. Collections of cnidarians for the AZGUprogram ceased last year on the basis that their chemical diversity was predictable and unpro-ductive in screens. Few hard corals have been collected given their low proportion of living tis-sue to inorganic skeleton, physical difficulty associated with making extracts, and conservationissues associated with collecting scleractinian corals.• Phylum Bryozoa . To date only a few of these species, known as moss animals or lace corals, havebeen collected, consisting only of 31 specimens in seven species. Although bryozoans are apotentially very productive group of sessile marine invertebrates, with a high proportion of bioac-tive compounds known worldwide, the tropical fauna contains species of generally small bodyweight, and with a low proportion of living tissue to inorganic skeleton, such that it is extremelydifficult to collect specimens of >100 g wet wt.• Phylum Chordata. In the same phylum as humans, there is a group of sessile animals called tuni-cates, ascidians, or sea squirts, that also have a demonstrated track record as being productivesources of bioactive compounds. During the AZGU program 397 specimens of ascidians, in 156species, have been collected, with an estimated 500 or more species living in Australian watersstill unsampled to date, many of which are important components to the encrusting and crypticcoral reef communities.Several other phyla, including mollusks, deep-sea fishes, crustaceans, and echinoderms have alsobeen collected during this program, with 324 specimens in 303 species sampled to date. By-and-large,however, these have not generally yielded any bioactivity of interest to the current program, and theircollection is more opportunistic, especially unusual species in unique habitats. Sponges, in particular, remain key targets for the AZGU biodiscovery program, providing a majorsource of new chemical structures and bioactive leads [5]. They are also the most interesting from a sci-entific perspective, containing a huge diversity of largely unnamed (new) taxa worldwide, with a par-ticularly rich and unique Australian fauna, and containing representatives of living fossils —taxa leftover from the Mesozoic era, apparently still alive and well. Since 1993, our knowledge of this sponge fauna has increased threefold, and more significantly,most of the new discoveries also represent species new to science. For example, in 1994 there were1385 species of sponges described for the entire Australian fauna (including its external territories),with less than half of these known to live in tropical waters [6]. This knowledge took 200 years toacquire. By comparison, in 2001 the published taxonomic knowledge base of Australian sponge diver-sity had grown to over 1500 described species, but more importantly, our collections now contain morethan 3000 species mainly from tropical waters, and most presumed to be new to science (i.e., they can-not currently be assigned to a published taxon). There has been a phenomenal advance in our marinebiodiversity knowledge of this phylum, which would not have been possible without this program.The formal processes of taxonomy (classifying and describing these species in the scientific lit-erature), however, will take a lot longer than the discovery of these species. Nevertheless, our ability todiscriminate these species as being new to science has already contributed significantly to marine bio-geography, biodiversity, and conservation. Several of these findings have already been published (or arecurrently in press). Three significant discoveries on marine fauna have arisen to date.Firstly, more than 600 species of sponges have been collected from the Great Barrier Reef (GBR)alone since 1993. Analysis of their taxonomic relationships [7], and ITS gene sequences of some keytaxa [8], suggest that there are at least two distinct sponge faunas within the GBR (far south and cen-tral-north), and possibly a third distinct fauna (far north, although not yet substantially sampled), all of which appear to have minimal genetic contact. These new data have important implications to the devel-opment of management and conservation strategies of these unique marine resources, which have usu-ally been assumed to represent a single GBR province. Similarly, from the same study, six distinctsponge faunas were discovered along the east coast, extending from Sydney to the Gulf of Carpentaria, R.J.QUINN et al.  ©2002 IUPAC, Pure and Applied Chemistry  74, 519–526522  each regional fauna containing many unique species (with a mean regional endemism of about 33 %)[7]. Previously, this entire east coast fauna was thought to consist only of two bioregions: a northern anda southern one. Secondly, the AZGU program also provided an opportunity in 1997 to revisit the historic site of the first comprehensive study ever made of coral reefs anywhere in the world, the Low Isles, off PortDouglas in Far North Queensland. These isles were the site of the 1928–1929 Great Barrier Reef Expedition, led by Sir Maurice Yonge, which conducted extensive experiments and sampling of thecoral reef biota. Much of our early knowledge of coral reef biodiversity, ecology, physiology, geology,etc., was derived from these early studies, and our own expedition was able to resurvey the sessilemarine invertebrate fauna, to gauge potential changes to biodiversity over time (e.g., influence of humanimpacts on these popular reefs) [9]. Thirdly, these marine sponge collections have been instrumental in delineating major biodiversity“hotspots” throughout tropical Australia. This study was possible by analyzing the extensive marinecollections using indicators of species richness, species uniqueness (endemism), and taxonomic affini-ties of these regional faunas in comparison with other regions in Southeast Asia and the western Pacific[10]. Based on a dataset of 2324 sponge species from 1343 investigated localities throughout tropicalAustralasia (which included datasets from studies pre-1993) 37 medium-scale (regional) faunas wererecognized, each distinct in some way from adjacent regions (e.g., different species richness, differenttaxonomic composition, etc.). Of these regions five were “hotspots” of species richness (>250 spp/ region): two located on the northeast coast (southern and northern outer GBR), two in the NorthernTerritory (east and west sides of the ‘Top End’), and one on the mid-northwest coast of WesternAustralia. Each of these regions contained about 27 % of unique species, not found in other regions. By combining some of the 37 medium-scale faunas according to their taxonomic affinities witheach other, further analysis identified 13 larger-scale faunas (domains), of which three had >600 spp(outer GBR, Top End of the Northern Territory, mid-northwest coast of Western Australia), and werefar richer than any other Australian marine province or domain, or the comparatively rich faunas of Vanuatu, Palau, and Thailand (which were used as outgroups for comparison). Each of these threeAustralian sponge “hotspots” contained distinctly different sponge faunas, with relatively few speciescommon to all domains (a mean endemism of 47 %). These three domain-sized “hotspots” representcompletely unique pools of genetic resources. Their existence also challenges our current simplisticmarine biogeographic models of a widespread, northern “Tethyan fauna”, with the erroneous presump-tion that these tropical genetic resources are homogeneous and widespread. New knowledge of the terrestrial flora Sixty plant species new to science have been discovered in the terrestrial flora collected since 1993.New populations of threatened species have been discovered in remote areas, providing the geneticmaterial which can be used to propagate the species. Record of weed encroachment in native forests hasbeen created for forest management use. Chemistry In an effort to find selective inhibitors of protein kinase enzymes, a marine sponge of the genus  Ircinia yielded three new cheilanthane sesterterpenoids as well as lintenolide ( 2 ) previously reported from thesponge Cacospongia cf. linteiformis [11]. Cheilanthane sesterterpenenoids have not previously beenreported from  Ircinia species. The compounds were found to inhibit MSK1 (mitogen and stress-acti-vated kinase) with an IC 50 of 4 µ M.In continuing efforts to develop selective adenosine receptor agonists, a series of natural productshave been discovered which display structure–activity within the various series. This information hasbeen of value in docking studies to understand the binding domains.  ©2002 IUPAC, Pure and Applied Chemistry  74, 519–526 Australian biodiversity via its plants and marine organisms  523
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