This overview describes a successful ICBG program established in Papua New Guinea (PNG) that is early in its second round of funding. The overarching goal of this ICBG reflects the original ICBG premise that human health and well being can be improved through an integrated set of programs dedicated to the description, assessment, rational utilization, and conservation of biodiversity in PNG. With more than 34 million hectares of closed tropical forests, PNG ranks ninth among the most forested tropical countries of the world.1 PNG is located in the Coral Triangle, site of the greatest concentration of marine biodiversity on Earth. We believe that the conservation and sustainable use of biological resources needs to be based on knowledge of the renewability of these resources, as well as an understanding of the socioeconomic pressures for resource consumption. Over 800 tribal groups inhabit PNG and many of them depend daily on local flora for medicines, food, housing and cultural materials. As noted in a Papua New Guinea Conservation Needs Assessment Synopsis Report, their interests compete with international economic pressures for the extraction of timber, fish, mineral and petroleum products.3 The PNG Department of Environment and Conservation (DEC) recognizes that a rational strategy for economic development in PNG must balance these pressures. Conservation of ecosystems is essential for the preservation of PNG's cultural heritage, and for future generations' legacy of PNG's unique and diverse biota. An understanding of the full range of biological resources existing in PNG is essential for their management, for the preservation of species critical to indigenous cultures, and for the development of strategies for sustainable use.
The central hypothesis of this work is that effective new therapeutics can be derived from the natural products of PNG's well-documented biological wealth. Such therapeutics, either marketed as Western medicines or as traditional phytomedicines, can provide needed revenue to local stake-holders and responsible PNG institutions. This income will provide alternative, “non-timber”, economic value to PNG forests, demonstrate value for reefs, and provide an incentive for the preservation of biological diversity. The PNG ICBG has been specifically designed to test this hypothesis. The timing of this project is particularly critical because of the accelerating rate of degradation of ecosystems in PNG, due in part to increasing human populations and the resultant impact on resources. This crisis was anticipated in the PNG Conservation Needs Assessment published in 1993 and has been confirmed in a new Geographic Information Systems (GIS) report published in 2008. This destruction of biological resources parallels a human cultural loss as oral traditions, intimately connected to their environs, are also lost. Fortunately, the pressures for unfettered development are being countered by governmental and non-governmental efforts directed at preservation of biodiversity and cultural traditions. PNG's unequalled, yet imperiled, biological and cultural diversity makes it a priority site for the integrated programs described here.
The drug discovery component of the current five years funding focuses on programs directed at human immunodeficiency virus (HIV) and tuberculosis (TB). The source organisms for drug discovery in the current grant period are terrestrial microbes and marine invertebrate animals. These collections will complement the plant based chemical diversity library that was created during the first grant period. The documentation of medicinal plant use in PNG continues to be an emphasis of this work. The medicinal plant surveys are complemented by projects conducted in support of the PNG Ministry of Health's Traditional Medicines Taskforce to provide chemical standardization and pharmacologic validation of medicinal plants that have been selected for development and promotion within the Taskforce program. The new Smithsonian Center for Tropical Forest Sciences/Smithsonian Institute Global Earth Observatories (CTFS/SIGEO) 50 hectare forest dynamics plot that has been established in the Ramu river valley, south of the Adelbert mountain range of PNG5 will serve as the center for conservation, botanical survey and biodiversity studies. This plot, herein referred to as the Wanang plot, is a unique and enduring conservation achievement that also provides a powerful research tool for studying forest dynamics, carbon sequestration and climate effects. This biodiversity resource will also permit integration of the botanical survey with studies of small plants and microbial diversity. The ICBG supported herbaria will play a pivotal role in the Wanang plot by facilitating the participation of PNG botanists in the plot inventory. The herbaria will also collect medicinal plants in support of the Traditional Medicines Taskforce.
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PNG is the world's largest and highest tropical island. The tropical forest of PNG is part of the third largest block of such forest in the world, with over 34 million hectares of closed canopy tropical forest. Though estimates of total world species vary dramatically, it is estimated that 300,000 to 700,000 different terrestrial species occur in PNG. This is close to 5% of all the world's species, even though PNG accounts for less than 1% of the world's land mass.6,7 It is estimated that 15,000 to 20,000 individual species of vascular plants may be found in PNG. Of these, perhaps 60% are endemic to Papuasia, the region including PNG, West Papua, the Solomons and Northern Australia. This is one of the highest rates of endemism in the world.8 PNG is home to some of the world's most unique species including the world's largest butterfly, moth, lizard, and crocodile. It is the home of the bird of paradise, a national symbol, crowned pigeons, cassowaries, echidnas, monotremes, dugongs and many other exotic species. An estimated 3,000 species of orchids can be found PNG. More species of orchids, sugar cane, parrot, pigeon and kingfisher exist in PNG than anywhere else on Earth. PNG is a ìcornucopia of ecologyì.
PNG's coastline includes 8,000 kilometers of mangrove swamps (part of the greatest extent in the world), lagoons, wet-lands, and coral reefs. The country's jurisdiction encompasses 800,000 square kilometers of ocean, including an estimated 40,000 square kilometers of coral reefs. PNG is part of the Coral Triangle, which encompasses over 50% of the world's coral reefs, and is home of the most diverse collection of species of coral, fish and marine invertebrates in the world.
In 1993, the Papua New Guinea Conservation Needs Assessment (CNA) synopsis report (see Appendix)3 was published by the PNG Department of Environment and Conservation with the Biodiversity Support Program (a US AID-funded consortium of the World Wildlife Fund, The Nature Conservancy and other conservation-oriented NGOs and local representation). The CNA reported to the government and citizens that significant degradation of ecosystems in PNG had occurred. A more recent GIS study4 reveals that this trend is continuing. The CNA report addressed land use issues, including communication among stake-holders, rights adjudication, modes of conflict resolution, and attitudes towards biodiversity. The CNA also synthesized available data into Consensus Maps of Biodiversity in PNG. The CNA maps provide an invaluable picture of the nation's biodiversity. Although there have been many expeditions to sample the flora and fauna of PNG, an estimated 20% of the country still remains unexplored in terms of biodiversity. This is illustrated in the CNA synthesis map #1 ìMajor Terrestrial Unknownsî.
PNG is a culturally diverse nation. Initially settled over 40,000 years ago, there are now more than 800 different languages spoken by an equal number of distinct tribal groups.10 Over 300 different dialects are spoken in the Sepik river basin alone.11 The lives of most of these tribal groups are intimately linked to their environment. They survive through subsistence farming, food gathering and hunting. Their food, medicine, housing, clothing and ritual materials are obtained from their immediate surroundings. This extended habitation of diverse environs by various tribal groups has led to an especially rich tradition of medicinal plant use in PNG. The decline of the environment has threatened the way of life for many of PNG's tribes.
PNG's economy struggled after gaining independence in 1975. Many of its emergent institutions, including the university, herbaria and national health systems, went under-funded. This economic situation is now improving, reflecting a renewed push by industrialized nations to partner in exploiting the extensive gold, gas, timber and fishery resources of PNG. Still, approximately 40% of the population lives on less than $2USD per day. The citizens of PNG suffer from the vicious cycle of poor health and poverty. Approximately one in 12 children dies before five years of age, and the overall life expectancy is less than 60 years. Access to Western health care for most of the population is limited to remote Aid Posts or regional Government Health Centers that are usually undersupplied. Thus, well over 50% of the population relies on traditional medicines for health care.
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In a proactive effort, the DEC established the PNG BioNET in recognition of PNG's developmental needs, its rich natural diversity, and the increasing threats to conservation of PNG's natural resources. PNG BioNET is an organization of PNG scientists and government officials, which serves the DEC in an advisory capacity on matters concerning the assessment, use, and development of PNG's biological resources. A complete description of PNG BioNET was provided in the 2003 application. Draft legislation, called the PINBio Act, seeks to establish PNG BioNET as the national clearinghouse for all research permits and access to PNG's genetic resources. Although the PINBio Act is currently “on hold”, a formal permitting procedure is in place through PNG BioNET and the DEC.
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HIV/AIDS continues to be a growing worldwide epidemic and is having a tremendous negative impact on PNG national health. HIV AIDS-related diseases are the major cause of death at the general hospital in the capital Port Moresby.13 According to AUSAid Centre for International Economics,14 PNG is on the verge of an epidemic that could infect over 25% of the population by 2020. PNG is the only Southern Pacific nation where AIDS is considered established. The situation calls for a concerted and comprehensive approach to the HIV/AIDS scourge. In this context, the contribution of indigenous herbal medicine that is widely utilized by rural communities needs to be examined and validated, in addition to implementation of Western alternatives. Infection with opportunistic pathogens and reactivation of latent infections, such as TB, are the hallmark of HIV/AIDS.15 Although the HIV genome and proteome are well described in molecular terms, the precise function of some of the viral proteins is poorly understood. This presents an opportunity for drug discovery to contribute to our understanding of the function of these proteins as new compounds that effect HIV are discovered. Also, new molecular targets will undoubtedly be discovered as new chemistry is tested for bioactivity. The majority of the HIV drugs currently in the clinic target reverse transcriptase or HIV protease, though drugs targeting HIV fusion and more recently HIV integration have been approved.16,17 Resistance to HIV monotherapy arises rapidly and anti-HIV therapy in developed countries is usually administered as a cocktail of three drugs of mixed molecular mechanisms.18 Standard therapy at the “Hedura (Help) Clinic” at Port Moresby General Hospital (PMGH) is usually a generic two-drug combination, although availability varies greatly, depending on contributions from international aid programs. In rural settings, patients are likely to receive a two or three week course of monotherapy, if even that is available (personal communication, Jackson Lauwo, Head of Pharmacy, UPNG). There is hope that a new AUSAid initiative will improve access to effective therapy, but the problem is immense.
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Tuberculosis is a communicable infection of lung and other tissue, caused by the bacterium Mycobacterium tuberculosis. The infection is commonly acquired by inhalation of airborne organisms produced by coughing. If the organism is not cleared, it slowly begins to multiply at the site of deposition. This multiplication triggers a cell-mediated inflammatory macrophage and T-cell response that contains the infection via the formation of granulomas. These granulomas contain viable bacteria in a dormant state, and correlate with immunity to subsequent infection and a positive TB skin test. In approximately 10% of infected individuals, the bacterial infection escapes the granuloma and triggers a destructive inflammation. The percentage is higher in immune-suppressed or malnourished individuals. The infection spreads throughout the lung, and eventually to other tissues. The disease is characterized by chronic inflammation, anorexia, low-grade fever and progresses to chronic chest pain and cough. In PNG, direct observed treatment short-course (DOTS) is an effective strategy for TB control when executed. About 55% of DOTS enrollees in PNG are considered cured, with approximately 20% (2005) of new cases under treatment. The World Health Organization (WHO) classified PNG as a country characterized by the highest level of infection. Treatment with current therapeutics can effectively decrease the morbidity and mortality of this disease, but drug resistant strains continue to emerge establishing the need for new treatments, particularly those with novel targets. The WHO estimates that one-third of the world's population is infected with TB, resulting in an estimated 2 million deaths each year. It is also estimated that approximately 30% of the population living with HIV are co-infected with TB.15 Additional data suggest that many of the deaths attributed to TB in PNG are in fact AIDS related.
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Malaria is a disease of protozoal infection characterized by high fever, hemolysis and liver failure. The infection is spread by an insect vector, the mosquito. Malaria is the third leading cause of hospital admissions and deaths in PNG. Malaria is endemic almost everywhere in the country and transmission is hard to control. Before large-scale control efforts began in the 1950s, parasite rates were 35% overall and 64% among children under 10 years of age. Unfortunately, in 1983 eradication efforts were abandoned when it became apparent that technical, operational, and administrative constraints precluded success. Malaria remains a serious health problem in coastal and inland regions. It is a constant threat up to an altitude of around 1200-1500 meters, where it becomes a periodic threat. Transmission is persistent throughout the year, with Plasmodium falciparum causing an estimated 75% of infections, with P. vivax a significant threat. Only the higher mountainous areas and Port Moresby are relatively malaria-free.
The number of confirmed cases of malaria peaked at 99,000 in 1995. Rates appeared to decrease to 20,900 in 1998, but this is now thought to be due to a reduced surveillance system rather than a real reduction. There were 81,192 confirmed cases in 2000, approximately 15.82/1000 population, with 617 deaths. Unfortunately, drug resistant malaria is detected in over 90% of the infections in some provinces of PNG. The need for accessible and effective antimalarial formulations at the village level is paramount. In the first round of funding, over 3,000 plant extracts were tested for antimalarial activity. Over 50 plant extracts with potent activity and relatively little cytotoxicity were identified. These plant activities are being pursued in the current round of funding, even though malaria screening has been discontinued due to its relatively high cost.
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Marine Organisms as Sources of Natural Products Chemical Diversity
The world's oceans cover greater than 70% of the Earth's surface, and taking into account volume, the oceans represent better than 95% of the biosphere. All but 2 of the 28 principal phyla in the animal kingdom are represented in aquatic environments; 8 phyla including the Coelenterata, Porifera, Bryozoa and Echinodermata are exclusively aquatic, largely from saline habitats. Greater than 95% of all animal species are invertebrates and conservative estimates are that there are over 1 million species represented in the world's oceans. It is generally accepted that invertebrates, and the animal kingdom in general, had their origins in the primordial oceans. Consequently, the animals that never left this environment have had a longer time period to adapt to their present environment. Also, because of the ability of sea water to moderate changes in salinity, pH and temperature, it can be argued that marine organisms live in a more uniform and stable environment than their terrestrial counterparts. Although it is difficult to provide unequivocal proof, conceptually it can be reasoned that marine invertebrate organisms, having propagated over eons in a more stable environment, have had the luxury of diverting greater amounts of resources to the development of secondary metabolic pathways as part of their chemical survival strategy.
The marine environment is also a very rich medium for the growth of microorganisms; the arguments stated above for development of unique metabolic pathways in marine animals are equally applicable to marine microbes. This is particularly true of microbes that are associated with invertebrate animals. There is mounting evidence that microbes develop very specific associations with marine animals and that this micro-environment can have profound effects on the secondary metabolism of the microorganism.
Marine natural products chemistry is a relatively young and undeveloped field of investigation compared to its terrestrial counterpart. The number of metabolites reported from marine organisms (animal, plant and microbes combined) is still quite small, less than 19,000, as compared to studies of plants and soil organisms that have yielded in excess of 150,000 compounds (based on analysis of the Chapman Hall Dictionary of Natural Products). Nonetheless, considerable progress has been made in the search for therapeutic agents from marine organisms in the past 15 years and two marine derived drugs, Prialt® and Yondelis®, have been approved for human use. To date most of the biological screening of marine natural products has been focused on cancer. There are currently fourteen marine candidates in clinical trials for treating cancer. Furthermore, there are several dozen other marine natural products which have shown very promising in vivo activity and are in advanced stages of preclinical development.
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Marine and Endophytic Microbes as Sources of Natural Products Chemical Diversity
As noted above, marine invertebrates are a rich source of novel chemistry, and they provide unique habitats for the proliferation of microbial communities. Even though symbiosis is a widespread phenomenon and an important agent of evolution, little is known about the symbiotic relationships between prokaryotic and eukaryotic taxa. The endobiotic environment is comprised of cells of microorganisms as well as the tissues of plants and animals that serve as hosts to a wide spectrum of microbial forms in a variety of relationships. Many of these associations began as incidental interactions that later developed into a relationship of obligate mutualistic symbiosis. This range of interdependence includes a wide spectrum of bacterial/host adaptations, including the biosynthesis and maintenance of unique secondary metabolites. Marine sessile invertebrates are considered a particularly rich source of novel metabolites and symbiotic microorganisms. Sponges, for example, may have up to 40% of their cellular volume occupied by associated bacteria. In many poriferans, complex microbial communities have been described that contribute to the lives of the hosts. In addition to providing a primary food source, microorganisms can also produce waste products, transfer nutrients, produce reef-like structures, or produce secondary metabolites.30, 31 It is widely believed that certain compounds and classes of chemicals are produced (or co-produced) by symbiotic bacteria. Numerous examples exist in which structurally related or identical compounds have been reported from taxonomically distinct marine invertebrates. Structural classes of natural products previously characterized from bacteria and more recently isolated from marine sponges and ascidians, lend support to the hypothesis that many marine invertebrate-derived natural products may be of microbial origin.32 Metagenomic analyses of the sponges Theonella swinhoei and Discodermia dissoluta have revealed that there are indeed bacterial symbionts responsible for the production of polyketides (PKS), nonribosomal peptides (NRPS), and hybrid PKS-NRPSs that have long been thought to be produced by only the invertebrate host.
Commensal marine bacteria inhabit surfaces, tissues, and internal spaces of marine macroorganisms, but the exact associations are transient and the interactions are not well understood. Since bacteria occur in seawater at concentrations of one million cells per milliliter, plants and animals are constantly exposed to high concentrations of bacteria. Many of these bacteria are motile, chemotactic, and/or pathogenic, and readily colonize a surface. Documentation of these co-existences is increasing and these bacterial films often play a vital role in the growth and development of marine plants and animals. These associations are significant in the quest for new bioactive natural products because of the potential cooperative role in the production of novel metabolites. These surfaces are richer in nutrients than seawater and most sediments, thus providing a unique niche for the isolation of many diverse microorganisms.
The terrestrial environment continues to provide much opportunity for the discovery of new metabolically productive bacteria and fungi. Genomic surveys proclaim that only a very small percentage of the microbial flora in soil have been cultured in the laboratory. Our focus in PNG is on the isolation of endophytic bacteria and fungi, which have emerged as excellent sources of new natural products.37 Endophytic microbes live within the tissues of plants without causing any visible harm to their hosts and can be isolated from surface-disinfected tissues. The colonization of a plant by an endophyte may be specific at the host-species level such as the relationship between fungi in the family Clavicipitaceae and several species of grasses and may even be specific to certain tissues. Endophytic fungi have been isolated from the roots, stems, and leaves of nearly every type of plant, including grasses, orchids, shrubs, and trees. In the tropics, where the diversity of plants, especially woody angiosperms, is richest, the biodiversity of fungal endophytes may be hyperdiverse. In contrast to endophytic fungi, the diversity of endophytic actinomycetes and their interactions with uncultivated plants remain poorly understood. However, evidence for the colonization of plants by actinomycetes other than the root nodule-forming Frankia spp. has increased over the past decade,41 and many of these isolates are proving to be prolific producers of secondary metabolites such as antibiotics, antitumor agents, plant growth promoters and plant growth inhibitors.38, 42 In addition, we have recognized that endophytic bacteria, particularly actinomycetes, are represented by many new taxonomic groups that have vast untapped biosynthetic potential. Endophytes may also be the source of novel compounds originally isolated from plants, such as paclitaxel44 and the antifungal agent 6-prenylindole.
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UPNG as the In-Country ICBG Partner Institution
The alliance with UPNG provides many advantages to the structure of this ICBG. It is the flagship University in the country and has a recognized administrative structure with established procedures for grant administration and program review and accountability. UPNG provides a nucleus of qualified collaborating in-country scientists dedicated to the goals of conservation, education, research, economic study, and drug discovery. UPNG faculties sustain collaborations with the FRI, which has provided considerable opportunities for this ICBG for type voucher maintenance and dissemination, and development of the Wanang plot. Developing the capacity of UPNG in its efforts in education, biological inventory, drug discovery and economic study are a means to develop infrastructure that will directly benefit all people of PNG. Enabling this powerful institution in areas relevant to this program will pay innumerable dividends in years to come. Furthermore, this collaboration enjoys the full support of university leadership, as evidenced by the signed MOU and letters of support.
PROGRAM LEADERS AND ASSOCIATED PROGRAM DESCRIPTIONS
The leadership skills of the Program Leaders have been critical to the success of this ICBG. Each program leader involved in this ICBG is an established independent investigator and is recognized internationally as an expert in their respective research field (Figure 1). Achievements of each Associated Program as well as collaborations among the leaders during the previous funding period are evident in the progress reports and publications.
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The associated programs of the PNG ICBG have been overwhelmingly successful in meeting their benchmark goals. The specific activities of each AP have synergized with those of other APs through interaction, data feedback, sample sharing and training projects to yield identification of new species, new pharmacological activities, new chemistry, publications and patent applications. The aims, rationale and approach of each AP are detailed in the individual sections later in this application. This current application builds upon the solid progress of the last funding period, retaining the synergies that have been developed while embracing the new emphases of the 2008 RFA. The minor changes in direction reflect progress made and the resulting evolution of short-term goals; as well as improvements in technology, procedures and interactions. Our proposed research and additional rationale for the major new directions in this application are described below.
We will continue the documentation of medicinal plant use in PNG by supporting the Traditional Medicines Database surveys and PNG student projects as part of AP 1's biodiversity research objectives. These activities will strengthen the Traditional Medicines Database as a national resource and provide powerful training opportunities for students in PNG. The UPNG herbarium will be supported to curate medicinal plant vouchers, and facilitate medicinal plant recollection as necessary. As indicated in the new specific aims, we will discontinue botanically-guided plant collections. AP 3 and 4 will participate in studies designed to assist in the validation and chemical standardization of medicinal plants selected by the PNG Traditional Medicines Taskforce for possible inclusion into the PNG healthcare formulary. Medicinal plants will be selected for study based on a comprehensive analysis of the Traditional Medicines Database and other resources.
There will be emphasis on novel microbes and marine invertebrates as sources for drug discovery during the next funding cycle. As noted in our progress report, the integration of microbes into our screening library resulted in the discovery of novel chemical entities with therapeutic potential. Wyeth is an international leader in the isolation of microbes from unique environmental niches, and has developed particular expertise with marine microbes, endophytic actinomycetes and endophytic fungi. Wyeth will focus on the annual isolation of approximately 200 new and phylogenetically distinct actinomycetes and fungi, from marine substrates and invertebrates, and terrestrial plants and soil. These isolates will be cultured under a variety of conditions, harvested, extracted and fully characterized by LCMS, UV/Vis, and ELSD. From these microbial isolates, AP 5 will produce approximately 2,000 HPLC generated fractions for industry- and university-based screening. Fungal and bacterial taxonomy and dereplication will be accomplished using a combination of techniques, including sequencing, morphology, conventional microscopy and electron microscopy. Phylogenetic assessment is performed by DNA sequence analysis as described in AP 5. Dr. Ireland's research group, in collaboration with AP 5 and Dr. Matainaho's group has collected marine invertebrates from various habitats in support of AP 5's marine microbiology program. The invertebrates themselves were not included in the ICBG in the first funding period due to budget constraints and the decision to focus chemistry efforts on plant extracts. Taxonomic examination of the marine invertebrate collections revealed that they are indeed remarkably diverse, contain many undescribed species, and new range records. Dr. John N.A. Hooper, Head of the Biodiversity and Geosciences Program, Queensland Museum, and world renowned sponge taxonomist is a long standing collaborator with Dr. Ireland. Dr. Hooper has participated in all of Dr. Ireland's sponge collection trips and will continue to do so in the future. He is working with Ms. Mary Kay Harper to publish assessments of marine sponge diversity in PNG and the taxonomic descriptions of new species. Chemical analyses of these taxonomically diverse sponges indicate that they are also chemically diverse. We have published a number of papers that clearly demonstrate novel chemistry and unique biological activities from PNG marine invertebrates.46 All marine collections were made with the full approval (prior informed consent) of local governments and under the jurisdiction of federally issued research permits coordinated by Dr. Matainaho. Starting with a collection of marine invertebrates from Kimbe Bay in 2007, AP 4 will contribute 200 marine invertebrates annually from a range of geographical locations and habitats within PNG. These organisms will be extracted and subjected to our HP20SS prefractionation protocol47 to generate 800 samples for industry- and university-based screening.
Tremendous progress has been made toward development of efficient screening protocols for analysis of plant and microbial samples during the initial phase of this ICBG. Our most productive target has been TB, yielding two patent applications, two provisional patent applications, and three manuscripts. Our pipeline for new anti-HIV agents is now open and several exciting leads are under investigation. Therefore, the university-based drug discovery effort of this application will focus on HIV and its most common life-threatening co-infection, TB. We propose to screen all microbial and invertebrate extract fractions for anti-HIV and anti-TB activity, and use cytotoxicity to eliminate false positives. We will use the improved university-based drug discovery assays described in the progress report for screening, followed by LCMS generated peak libraries for rapid dereplication and to facilitate bioassay-guided isolation of active metabolites. We will further evaluate active metabolites for potency, selectivity and establish an exact chemical structure, using state-of-the-art pharmacological and structural analysis techniques described in AP 3 and AP 4, respectively. We will analyze, store and upload all data to the GDC.
Industry-based drug discovery will focus on a parasitic animal disease panel that targets anthelmintic and anti-arthropod drugs. The assays are performed at the Fort Dodge facility of Wyeth Research, and are described in greater depth in AP 5. They include a C. elegans - based anthelmintic primary assay performed in culture. Hits in this screen are tested in a secondary in vivo assay using rodents infected with a parasitic nematode, followed by a tertiary sheep assay for advancement of promising rodent actives. Anti-arthropod drugs are detected using a parasitic larvae insect assay in 128-well format. Hits in this primary assay are followed in flea and tick contact assays, performed in glass vials. Tertiary assays include a flea ingestion assay to determine the quality of arthropod control. Wyeth Research is currently reorganizing its HTS drug discovery strategy; in the interim samples will be screened in assays being operated in individual programs in the therapeutic areas of oncology, neurosciences, cardiovascular, inflammation, and women's health. These in-house phenotypic assays are Wyeth's secondary assays that are used to confirm HTS. Implementation of HTS will be pursued by AP 5 following the reorganization process.
This application proposes to build on our experience with training UPNG students. In this next funding period we will host two to four UPNG graduate students, for one-year sabbaticals at the University of Utah, to train in pharmacology and medicinal chemistry. An extended training sabbatical is planned for Ms. Beatrix Waiin, who has an M.S. in Microbiology and is a faculty member at UPNG, at Wyeth Research to gain experience in the most modern techniques for microbe isolation and identification. The ICBG will continue to support professional development of collaborating scientists and offer them opportunities to present their current research at international meetings. Through workshops, technology transfer, and development of infrastructure, the ICBG will continue to develop the capacity of laboratories at UPNG and FRI to conduct innovative research that directly supports the goals of this ICBG.
Outreach, intellectual property rights and access and benefit sharing activities will continue in the next grant cycle, starting with input from researchers at the University of Utah, the Smithsonian Institution, the Binatang Center and the Institute for Medical Research on aspects of the PINBio Act that will directly affect international collaborative research that accesses PNG genetic resources.
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Research Plan Progress Report years 1-5
The Specific Aims listed below are stated verbatim from the original application funded in 2003. An overview of progress is provided here. Detailed discussions of progress and proposed research are described in the individual AP sections.
Specific Aim 1: Support Herbaria at UPNG and the FRI for the documentation and monitoring of plant diversity, and for the provision of long-term data on the abundance, distribution and dynamics of floral and faunal species. To inventory, enumerate, identify, and describe plant species found in PNG. To use these studies as training opportunities for forestry, botany and land management by conducting workshops and by the training of participating individuals. To collect plants samples for pharmacologic evaluation.
Specific Aim 2: To expand existing Herbaria at UPNG and the FRI for inclusion of vouchers prepared in conjunction with the botanical inventory, socioeconomic valuation, and ethnomedicinal studies. To adopt the Natural Products Information System (NAPIS) for the management of extracts and their fractions, and for their prompt distribution to the appropriate associated programs, and for recollection and re-extraction. To establish a "gene bank" for preservation of floral genetic material, cross-referenced to herbaria vouchers and suitable for future amplification and marketing and development for scientific and industrial exploration.
The botanical surveys conducted by the Herbaria targeted PNG indigenous or endemic species as part of a country-wide survey. This activity linked the collection of field vouchers for taxonomy with collection of samples for subsequent chemical and pharmacological analysis. Trained botanists directed the ecologically sensitive collection of approximately 1 kg of plant parts along with field pressed vouchers. All plant samples were labeled with herbarium collection/accession numbers and subcodes, recorded in the NAPIS database, dried and stored at UPNG or FRI for milling and for future use.
Year 1-5 botanical collections focused on poorly documented lower level and coastal rainforests on the main island of PNG and surrounding islands. The current plant collection total is 728 species (over 1,300 individual plant parts), representing approximately 500 genera and 300 families. This unique collection documents largely unknown distribution and varieties of PNG flora. PNG herbaria have distributed over 1,700 type specimen vouchers, representing 660 species, to recognized herbaria including the Royal Botanic Gardens, Kew Palace, London; Nederland National Herbarium, Leiden University; Harvard University Herbaria, Boston; The Smithsonian Institution, Wash. D. C., and others in Asia and Australia.
Several new species have been tentatively identified including the first collection and report of the genus Tapeinochilos from the D'Entrecasteux Islands. Other significant discoveries from the D'Entrecasteux Islands collections include a number of distinct species currently classified as Chisocheton seyeri, as well as two potentially new species, a Zingiberaceae, Riedelia sp. (Fig. 3), and an Anonnaceae. Other likely new species are being evaluated.
Specific Aim 3: To repatriate species identification, population, distribution and ethnological data obtained from major PNG collections that have been distributed globally, beginning with the Smithsonian Institution and the British Museum of Natural History.
Progress: Taxonomic information on PNG insects and plants has been made available to the PNG scientific community through digitization of specimens deposited outside the country. The focus of these efforts has been on herbivorous Lepidoptera and their host plants in connection with the ecological studies of the Binatang Research Center, Dr. Scott Miller at the Smithsonian, and Dr. George Weiblen at the University of Minnesota. Dr. Miller has overseen the digitization of type specimens of PNG moths (especially Geometridae) in order to accelerate taxonomic progress.48 These efforts are tied to DNA barcoding49 and dissemination of images on the web.50 Dr. Weiblen and colleagues have developed an online checklist of PNG flora51 and a web-based repository for plant collection data and digital images (http://atrium.ng.brit.org/). Under the direction of Dr. Weiblen (AP 2) and colleagues, the Binatang Research Center mapped, measured, and identified all woody stems >5 cm in diameter in two hectares of lowland tropical rainforest at Wanang, PNG. These activities provided an estimate of the Wanang plot woody plant diversity including at least 70 families and 500 species. These baseline data will accelerate inventory of the plot and subsequent plant taxonomic discovery.
Specific Aim 4: To develop and conduct ethnomedicinal surveys in order to identify pharmacologically active plants and to evaluate those used for fever or cough for anti-malarial and anti-TB activity. To collect and preserve cultural information concerning the use of traditional medicinal plants. To promote organization and empowerment of local stake-holders on the local, regional and national scale. And, to provide mechanisms to ensure access to data collected and to royalties and resources returned to participating institutions, communities and healers. To involve PNG participants in all ICBG programs, especially those involving the collecting of samples, interviewing local populations, bioconservation and drug discovery.
Progress: The documentation of medicinal plant use and the preservation of associated traditional knowledge via the Traditional Medicines Survey have been very successful. PNG ICBG reinvigorated this project, which now trains and annually supports an average of four senior pharmacy students to administer the WHO approved survey questionnaire. The students are trained in questionnaire administration, traditional medicines use, taxonomic nomenclature, herbarium specimen preparation, and instructed in the preservation and documentation of traditional knowledge and culture. Students are also supported for a one year elective that includes travel and living costs. Written reports from these students are sophisticated, and contain analyses of interviews with many traditional healers, pictures and documentation of the plants, their preparation and use. As a result of ICBG support, the Traditional Medicines Database contains information concerning over 1,500 traditional medicines, and is now viewed as a national resource (see National Policy on Traditional Medicine). Legislation is under development that will recognize the intellectual property of the contributing healers and establish benefit sharing compensation schedules for outside access to the database. The Database is employed to guide the selection of traditional medicines for chemical and biological evaluation by the Traditional Medicines Taskforce, chaired by Dr. Rai of AP 1.
Specific Aim 5: To develop and conduct economic value assessment of biological resources in order to quantify the economic value of biological resources for comparison with other resource use options. To identify production and marketing of biological resources in local markets, that provides income for local residents, in order to identify economically important plants. To list, describe and collect vouchers of flora species used for nutrition and cultural purposes.
Progress: Dr. Billy Manoka has developed a field assessment tool for economic evaluation of biodiversity conservation of ICBG target sites. The survey was pre-tested on households from Urumarav village in the Almami area, Madang Province in 2006 and 2007. The primary objective of the survey was to determine the local communityîs willingness to support the protection of their rich terrestrial biodiversity. The survey specifically identified cash income activities and generated an economic value for biodiversity from the communityîs perspective.
Specific Aim 6:
To screen approximately 1,000 plant extracts yearly for pharmacologic activity. To code and reference each sample to corresponding herbarium vouchers. To fractionate, dereplicate and identify lead molecules in each of the target disease areas. To initiate advanced preclinical evaluation of lead molecules, concerning molecular mechanism of action, pharmacokinetics and toxicity. To transfer basic technology for plant extraction, extract fractionation and pharmacologic evaluation to UPNG. To conduct workshops at UPNG, and to host UPNG scientists at University of Utah and BYU for training experiences. To provide 1,000 samples per year to Wyeth Research (AP 5) for comprehensive pharmacologic evaluation in industrial HTP screening. Two perform pilot toxicity studies of PNG natural products with drug potential for the initial estimation of their therapeutic index.
Progress: The extraction and prefractionation protocol used was developed to facilitate rapid chemical and pharmacologic analysis and dereplication of metabolites in plant extracts. Our laboratories have made several innovations to a prefractionation procedure that yields samples suitable for LCMS dereplication and are compatible with screening formats. These new methodologies have been presented at national meetings, published in the peer reviewed literature, and are described in AP 4. The prefractionation procedure effectively concentrates active compounds, thereby increasing the likelihood of their detection in bioactivity screens. Over 3,100 prefractionated samples have been generated from our botanical collections, formatted into 96-well plates, and distributed for screening. Prefractionated samples identified as hits in the bioassays are now routinely separated by HPLC into a 96-well plate (20 fractions per sample), in parallel with High Resolution Electro-Spray Ionization (HRESI) mass spectrometry for rapid dereplication and identification of active natural products.
HPLC affords a second dimension of chromatography that very successfully separates extract components throughout the liquid chromatogram profile and usually yields subfractions containing only one or two compounds with determined accurate mass values. Greater than 99% of the sample is directed to a fraction collector, while a small portion is split to a Micromass Q-Tof micro equipped with lockspray for accurate mass measurements. The fraction collector distributes fractions into 96-well plates suitable for further bioactivity testing. When active wells are identified, a mapping function is utilized to identify MS peaks that correlate with activity. Analysis of the maps easily identifies background ions, and ions from the bioactive wells can be extracted. The accuracy (-5 ppm) is sufficient to identify molecular formulas that are used for rapid dereplication in conjunction with taxonomy. This LCMS protocol represents a major technological advance in natural products drug discovery. Obtained mass values allow identification of known compounds, while the subfractions are perfectly suited for confirmatory bioassay.
During the first funding period, over 3,000 botanical fractions and 2,500 microbial fractions were analyzed by AP3 in each of the TB, HIV, malaria assays and cytotoxicity counter screen. The university-based drug discovery program employed whole pathogen drug discovery screens for TB, malaria, HIV, and T-cell cytotoxicity. The cytotoxicity assay was useful to identify plant fractions containing cytotoxic agents with potential as anti-cancer agents and proved useful as a counterpoint to the infectious disease screens, thus enabling identification of non-cytotoxic anti-infective agents. To meet the demand for throughput, we have established drug discovery assays in a uniform format for extract screening and data analysis. All of our screens are BSL-2 level containment and are performed in 96-well plates. Data are exported directly from plate readers into EXCEL© spreadsheets that have been customized to automatically calculate results for each assay. The spreadsheets also generate formatted data that can be directly uploaded into the NAPIS© GDC database. AP 3 also employs Clinical and Laboratory Standards Institute (CLSI) established protocols to establish MICs of TB active metabolites against a panel of bacteria, which helps assess selectivity.
AP 3 has completed over 18,000 assays with plant extracts, yielding 636 non-toxic hits in the infectious disease panel. Non-toxic hits are defined as active fractions yielding less than 30% growth inhibition of human T-cells in culture. The HIV screen identified 340 non-toxic hits that exhibited T-cell protection comparable to AZT (out of 6,933 samples screened). Malaria and TB hits are defined as those fractions showing >70% inhibition of malaria or TB growth. For malaria the total of non-toxic hits was 132 out of 3,397 assays, and for TB the total number of non-toxic hits was 164 out of 3,555 assays (data are detailed in AP 3). These hits were subjected to dereplication and confirmation using secondary assays, which led to selection of projects for further study in AP 4. These studies identified a number PNG plants with exciting potential. For instance, Coelospermum salomoniense has yielded a known anthraquinone with potent anti-TB and anti-malarial activity.52,53 Rhus taitensis yielded a new metabolite, tetrahydroxysqualene, with potent anti-TB activity. Exocarpus latifolius yielded exocarpic acid54 and several new derivatives, which we have shown to have potent and selective anti-TB activity. The exceptional selectivity of exocarpic acid and tetrahydroxysqualene has led to provisional patents currently being created at the University of Utah Technology Commercialization Office. Fractions from Planchonia papuana, harvested for timber in PNG, demonstrated potent anti-HIV activity.55 In fact, several trees in our collection that are harvested for timber show verifiable activity against TB, HIV or malaria. Finding non-timber value may be an important impetus for forest preservation and future botanical exploration. These studies are detailed in the progress reports for AP 3 and 4.
In years 1-5 Wyethís development of isolation and cultivation techniques has been highly successful. A total of 948 purified microbial strains from PNG plant and marine substrates were subjected to dereplication by ribotyping, ribosomal gene sequence comparisons, and morphology. As a result, 536 strains were determined to be genetically and/or biochemically unique. A highly effective process was developed for the isolation of endophytic actinomycetes from a variety of plant tissues. Remarkably, these procedures resulted in the isolation and characterization of at least five potentially new genera of plant-associated actinomycetes. For inclusion in high-throughput screening at Wyeth, 381 unique microbes were cultivated in several different media to generate 4191 pre-fractionated samples. Industry-based HTS (AP 5) of ICBG plant and microbial samples exceeded 75,000 separate assays in more than 30 screens, including anti-infectives, cancer, inflammation, womenís health, neurosciences, and metabolic diseases.
During the previous funding period, AP 3 screening procedures were optimized to increase capacity. The improved throughput allowed screening of Wyethís ICBG fractionated microbial library in the AP 3 assays. Initially, we tested 2,500 fractions from Wyethís microbial isolates in TB, our highest throughput assay. These TB actives were further tested for activity against two Gramñ and three Gram+ bacterial strains using CLSI protocols.56 Of the 65 TB hits, 13 were TB selective and were not toxic to any of the other five bacterial strains. On the other hand, eight were active against all of the five additional strains, three of which exhibited no cytotoxicity to human cells in culture. On the basis of taxonomy and chemistry of the producing strains, prioritized hits were fractionated at Wyeth and guided by activity from AP 3 assays. Several rounds of this interaction resulted in Wyeth initiating patent filings in 2008 on the first two new antibiotics from ICBG isolates. An analysis of the microbial library in HIV and malaria was also productive. For HIV a final set of five highly active fractions were identified, two being as active as our AZT control. For malaria, 33 extracts, mostly from actinomycetes were confirmed active, but several were cytotoxic and not pursued. This exchange of material and data between an academic and industrial partner is extremely innovative and exemplifies the true collaborative and cooperative nature of this ICBG.
This ICBG has embraced the idea of professional development for collaborating scientists and students, and has made training at all levels a priority. We believe that the longest lasting impact the ICBG can make is through the education of up-and-coming scientists dedicated to the goals of scientific advancement, conservation and social progress. Project Leaders from PNG have been supported to attend international professional meetings including the American Society of Pharmacognosy, Society for Economic Botany and a meeting on Taxonomy of Melanesian Plants at the Netherlands National Herbarium. Workshops in the areas of endophyte isolation, TB and other microbial screening, and database management and questionnaire administration have contributed to student training. In addition, the continued development of herbaria and laboratory facilities has made ICBG related student projects in PNG feasible.
One of the great accomplishments of this ICBG has been student training. Approximately 33 undergraduate and graduate students have been supported at UPNG, with over 20 gaining diplomas. ICBG-supported student Masterís and Honorís projects have focused mostly on plants used as traditional medicines. Three of these students were hosted at the University of Utah for the laboratory research portion of their theses. They were trained in rigorous scientific methods, conducted advanced studies on traditional medicines, and generated publishable results. The first publication from this training partnership was the study of Evodia (Melicope) elleryna, used in the Kurti region of Manus Island for cough and fever.57 The second reported a new anti-TB natural product from a tree that is harvested for timber in PNG.58 Two trainees that studied at the University of Utah, Ms. Emma Powan and Ms. Manah Dindi, have obtained their Masterís degrees, and now hold lectureships at UPNG. Mr. Jeffrey Noro has completed requirements for a Masterís degree, which should be awarded shortly. An additional student completed her Masterís degree working on an ICBG project with Dr. Rai and is now a lecturer at a UPNG satellite campus. Ms. Powan and Mr. Noro have been accepted into Ph.D. programs starting next year at the University of Auckland and the University of New South Wales, respectively. Mr. Owen Paiva, a current Masterís student, has been accepted into the Ph.D. program at the University of Queensland, pending receipt of his degree. There are four additional students supported by ICBG funds currently enrolled in Masters programs at UPNG. In addition, the ICBG has provided research opportunities for 13 University of Utah students and postdoctoral fellows. Four of these students have finished Ph.D. degrees and are currently postdoctoral fellows.
Specific Aim 7:
To select certain plants, based on pharmacologic, ethnobotanical, economic and ecological considerations, for potential development into standardized herbal preparations in future years. To develop analytical methods for standardization of the active components of these potential phytomedicines and to perform pilot toxicology studies on the preparations. These phytomedicines would target local and domestic markets as affordable remedies, while partnerships for export to international markets will be explored as well. To explore in-country mechanisms for establishment of production and marketing. Income will be channeled back to local communities via private industry and mechanisms established above.
Progress: In 2007, the government of PNG adopted a National Policy on Traditional Medicines. The policy identifies specific pathways for the development, promotion and integration of traditional medicines into the National Healthcare System. A Traditional Medicines Taskforce has been appointed, with Dr. Rai (Project Leader in AP 1) to head the taskforce. The taskforce is charged with promoting the policy nationwide, selection of safe and effective traditional medicines for development and incorporation into the nationís healthcare formulary, the preparation of a training manual for traditional practitioners in primary care, and the outreach to and organization of traditional healers nation wide. The Traditional Medicines Health Care Initiative provided a unique opportunity for the PNG ICBG to contribute directly to the improved health status of PNGís population.
Dr. Rai and the other members of PNG Traditional Medicines Taskforce use the information contained in the Traditional Medicines Database to prioritize selection of traditional plants for in-depth study. The first selection of the Traditional Medicines Taskforce for was a topical analgesic and anti-inflammatory preparation made from species of Parmeliaceae lichen. Work performed in AP 4 (see progress report) showed that approximately 95% of a hexane extract of the lichen was composed of the orsellinic acid derivatives, atranorin and chloroatranorin. These compounds have characteristic UV absorption spectra and will serve perfectly as standards to validate batch-to-batch consistency. Work in AP 3 showed that high concentrations of atranorins inhibit COX 1 and, marginally COX 2. Additional traditional medicines chosen by the Traditional Medicines Taskforce are under investigation.
Specific Aim 8: To maintain Memoranda of Understanding (MOU) among the institutions central to this ICBG. To establish and agree to formulae for benefit sharing and Intellectual Property Rights (IPR) safeguarding. To serve as an international example of collaboration, making progress toward the long term preservation of PNG biodiversity, while at the same time directly benefiting the people of PNG.
Progress: The MOU and consortium agreements signed among the University of Utah, UPNG and Wyeth embody the ideals of the Convention on Biodiversity and were designed with legal counsel from each institution and PNG BioNET. These documents resulted in development of a permitting procedure for scientific research projects involving PNGís genetic resources, which requires approval at national, provincial, and local government levels. Our Prior Informed Consent and Access and Benefit Sharing schedule govern interaction with local communities. These protocols were accepted by the Fogarty International Center in 2004. The protocols have become our standard operating procedure (SOP) for reconnaissance, collecting and appreciation visits between herbaria staff, other participants, and the collaborating communities. The benefit schedule includes visits from the Regional Medical Officers when possible. Figure 4 is a picture taken at an ICBG sponsored medical clinic on Bougainville in 2006. Our MOU and SOP have been formally adopted by the DEC as prototypes for new research applications concerning PNG genetic resources. As a mechanism to further formal national legislation, the ICBG has commissioned two formal reviews of PNG government approved research agreements and policies concerning Prior Informed Consent, Intellectual Property, and Benefit Sharing along with two formal workshops on the same topics. These efforts have contributed to the pending íPINBio Actî legislation, concerning compensation for access to intellectual property and genetic resources, and for the development of traditional medicines.
Outreach by ICBG projects has taken several forms. Meetings with provincial and local level governments and collaborating communities are part of our reconnaissance and appreciation visits. Traditional Medicine Taskforce outreach includes regular meetings with provincial officials and tribal groups of traditional healers. ICBG annual meetings in PNG arrange for attendance by representatives from collaborating communities and other government agencies. The ICBG has also co-sponsored the annual PNG Medical Symposium and presented program descriptions there.
The original five-year MOU governing this work has recently been renewed after review by UPNG administration and legal council (the new signature page attached to the end of the original MOU in the Appendix). A consortium agreement was signed in 2003 between Wyeth Research and the University of Utah recognizing this MOU. MTAs have been signed between the University of Utah, the NIAIDís TAACF and the Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago.
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We have established an immensely successful set of integrated programs that promote biological resource survey, conservation, drug discovery, technology transfer and education in PNG. These goals were achieved by empowering PNG institutions and researchers and linking them with capabilities found at the University of Utah, the Simthsonian Institution and Wyeth Research. The work has required the parallel development and acceptance of policy initiatives governing research on PNG genetic resources and intellectual property, and approval of protocols for prior informed consent and equitable benefit sharing. The activities of the associated programs have synergized to yield successful description of new microbial and botanical species, new pharmacological activities, new chemistry, publications and patent applications. The collaboration has provided technology transfer and training opportunities for our partners in PNG and is documenting non-timber value of rainforest biodiversity. Advancements in chemistry and screening have yielded extraction, fractionation, screening and data management protocols that have greatly streamlined the identification of new chemistry and medicinal plant activity relevant to human health.
Drug discovery efforts of this new application will focus on the chemical and pharmacological analysis of PNG microorganisms and marine invertebrates. University-based work will primarily target anti-HIV and anti-TB activity. In this application, only selected medicinal plants of interest to the Traditional Medicines Taskforce or student projects are emphasized botanical sources. Industry-based drug discovery will focus primarily on parasitic diseases and animal health. The proposed research will take advantage of the technical advancements from the previous funding period to accelerate the identification and development of new molecules and ethnomedicinal plants with therapeutic potential in human health.
ICBG activity has resulted in new molecules from PNG biodiversity moving into advanced stages of preclinical development, including the initiation of four pending or provisional patent applications mentioned earlier. This exemplifies our first pathway for drug development. Other collaborations add value to university-based projects. Under signed MTAs, three anti-TB compounds are being tested for further development in the TAACF and Institute for Tuberculosis Research, University of Illinois at Chicago's in vitro models of quiescent TB disease. Candidates that show promise will be taken into animal models and further developed. Further development of medicinal plants or traditional medicines will follow the pathways prescribed by the PNG National Policy on Traditional Medicines and legislation pending approval in the PNG parliament. Further development of medicinal plants will likely begin in-country, with information dissemination and testing within PNG under the supervision of the Traditional Medicines Taskforce.
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