Final Report Summary - NATPHARMA (Reinforcement of research potential for the realisation of a complete drug development scheme from natural compounds)
Executive Summary: The result expected from NatPharma was the growth of the research potential of the NeaNAT group. This goal has been achieved, and the most significant results obtained are: i) the research facilities, ii) the human potential, and iii) the connections with other research institutions and with the local productive infrastructures. The NeaNAT group now owns state-of-art equipment for molecular biology, which is a necessary requirement to exploit the new expertise acquired through the NatPharma project, and in addition it can attract researchers from other groups to visit us and start collaborative projects. The analytical capacities of the NeaNAT group are remarkably increased by the Orbitrap high-resolution mass spectrometer, which will be used by all the researchers of the NeaNAT group, and will allow the group to provide analytical services to private companies and environmental monitoring services to local, national and European public organizations. The NeaNAT group also gained a powerful hardware and software computational infrastructure, that will be the basis of the work of the group in the next years. The human potential resulted greatly improved by the recruitment of two researchers expert, respectively, in the identification and expression of biosynthetic genes and in quantum-mechanical calculations for interactions of drug candidates involving metals. The recruited researchers worked in close contact with staff members, leading to an effective training of the whole NeaNAT staff, so that the required knowledge is now permanently acquired by the group, even though external conditions made impossible the permanent recruitment of new researchers. The ability to study the biogenetic pathway of a natural product is essential to keep NeaNAT in an outstanding position in its field. Expertise in the expression of biosynthetic pathways can be used to create easily-cultivable bacterial strains producing useful natural products, strongly increasing the chance of success in the development of drugs from natural products, and leading to collaboration with pharmaceutical industries interested to exploit the potential of marine natural products. The application of computational methods in drug design is greatly improved by the possibility to integrate quantum mechanical simulations, making it possible to challenge wide-ranging issues in medicinal chemistry and providing new tools to improve the design process. Since a large number of molecular targets of pharmaceutical interest are enzymes catalyzing a metallo-mediated reaction, the acquired knowledge will allow us to renew the academic collaborations and industrial research agreements already gained during the project, as well as to establish new research platforms either with academic or industrial partners. The strengthening of NeaNAT in terms of human potential and of equipment have already shown some results. The group is part of the European-Chinese Research Staff Exchange Cluster for Marine Biotechnology (call FP7-PEOPLE-IRSES), financed by the EC (Grant agreement no. 246987) in August 2010 (two Chinese researcher are currently working in our group), and applied, in a consortium of 16 research organizations and enterprises, to the call FP7-KBBE-2012-6 with the integrating project "BlueGenics", a project that fits perfectly the theme of NatPharma. The new expertise in drug design of the group led to two research contract with Sigma-Tau Research Switzerland S.A. Thanks to NatPharma, NeaNAT might also contribute to the regional development. The group has reached a four-year agreement (2011-2015) with the Campania region to monitor the possible presence of toxins produced by blooms of the alga Ostreopsis ovata, and will try to establish a closer connection with research organizations and biotechnological companies based in Campania. The Campania region launched for the realization of a network for biotechnology financed through structural funds FESR 2007-2013, and NeaNAT will apply to be one of the actors in this network. Project Context and Objectives: The aim of NatPharma was to bring new scientific knowledge to the NeaNAT group, a multidisciplinary research group based at the Dipartimento di Chimica delle Sostanze Naturali, University of Naples "Federico II", which has been active for over 40 years in the isolation and identification of natural products of marine origin, expanding along the years this core activity to the study of the biological and pharmacological activity of the isolated compounds and of their biosynthesis. The object was to provide the group with all the capacities for the successful realisation of a complete drug development scheme from natural sources to optimized drugs, including the identification, the clarification of the mechanism of action, the optimization and the bio¬technological production of bioactive compounds from marine sources. In particular, in order to enhance our ability to develop new drugs from natural products, the NeaNAT group needed to integrate the pre-existent skills in the natural product chemistry and in computa¬tiona¬l methods for drug design by acquiring new expertise in the fields of: i) biotechnology for the identification of genes in¬volved in metabolic pathways leading to the biosyn¬thesis of bioactive compounds and ii) quantum-mechani¬cal calculations (ab-initio methods) for the investigation of the interactions occurring between organic molecules (drug candidates) and their biological targets. NatPharma addressed the aim of this proposal adopting a multilevel strategy for acquiring the required expertise and for the growing of knowledge of the whole group, by means of a) two-way secondment of staff researchers, b) recruitment of expert researchers, c) participation at workshops and courses and organization of seminars, and d) dissemination activities. These actions were integrated by a major upgrade of research equipment related to the study of biosynthetic pathways and quantum-mechanical calculations. Project Results: The project has developed according the plans, with only minor changes. The two new area of expertise we intended to cover thanks to the project are : i) biotechnology and ii) quantum-mechanical calculation. Therefore the purchase of new equipment was directed to the improve the molecular biology lab and the computational chemistry lab. The purpose of new equipment was to improve the throughput of the molecular biology laboratory, and to guarantee an efficient and performance-optimized computational laboratory suited for ab-initio calculations. These goals were achieved in the estimated time. Some changes were made in the instruments acquired compared to the original plan. We decided not to buy a colony picker, because the developments in the screening strategies make this instrument by far less useful than initially estimated. Therefore, we evaluated that a more profitable way to increase the throughput of the lab was to buy several smaller state-of-art instruments. We also needed to perform some small infrastructure work at the molecular biology lab to make room for all these new instruments and put them in the best working conditions. The result is a molecular biology laboratory mostly equipped with brand-new and efficient equipment. We also purchased a top-level high-resolution mass spectrometer with much better performance than usual triple-quadrupole LC-MS instruments, the Thermo Orbitrap XL. A high-resolution LC-MS is more valuable than a standard LC-MS for our work because, while keeping the extremely high sensitivity of any mass spectrometer, it allows the determination of the molecular formula of the compounds analyzed in addition to their molecular mass, making their identification far faster and easier. This instrument strongly improves the analytical capabilities of the group. To guarantee an efficient and performance optimized computational laboratory suited for ab-initio calculations, we needed to perform some infrastructure work and by purchase a high performance computer (HPCC) system, graphical workstations, and ab-initio software licenses. The computational laboratory is based in three different rooms, so it was necessary to design and implement a optical-fiber based network and to air-condition the room hosting the HPPC system. The hardware for the high performance computing was chosen considering the need to have the best ratio between occupied space and calculation power. A Gigabit-Ethernet based High-Performance Computing Cluster was built, with 6 Twin servers 1U, for a total of 12 nodes, a RAID storage system with an effective capacity of 4 Tb (4 Tb in), and a 10KVA UPS. The choice of the software, done on the basis of an in depth analysis of recent literature about ab-initio studies on interactions between transition metal and organic compounds, led us to choose Gaussian 09, and due to an agreement between our University and the company Gaussian Inc., we were able to receive for free a full license for the use of the ab-initio software Gaussian 09 and its graphical interface Gaussview. Finally, in order to guarantee an efficient and performing optimized computational laboratory, it was necessary to integrate the operating system of the newly acquired Linux based computers with existing Unix based workstations. Two experts in biogenetic pathways (Dr. Thomas Hochmuth) and ab-initio methods (Dr. Francesca Rondinelli), both coming from leading research groups in the respective fields, were recruited with two-year contracts. Dr. Thomas Hochmuth worked under the scientific supervision of Prof. Alfonso Mangoni. The main goals of his recruitment was to strengthen molecular biology at the NeaNAT. He first arranged and organized the new equipment, and built contacts to suppliers of materials and a good sequencing service. Then he started the actual scientific work and the transfer of expertise to the group, which was performed both in the day-by-day work and in the group meetings, by a continuous training of other researchers in the group particularly of Dr. Gerardo Della Sala and Dr. Roberta Teta. Dr. Hochmuth identified several PKS genes from the metagenome of P. simplex, and introduced in the NeaNAt group the techniques necessary for heterologous expression of these genes. In general terms, this two-year recruitment provided the NeaNAT group with all the expertise needed to work on the study of the biosynthetic pathways, and with the basic expertise needed to work on heterologous expression. Unfortunately, while we were very satisfied with the work of Dr. Hochmuth, we were not be able to offer to Dr. Hochmuth a permanent position or at least a further contract in line with his expectations, so he chose to leave the group. In spite of this, the expertise provided by Dr. Hochmuth is now acquired by the group and particularly by Dr. Gerardo Della Sala and Dr. Roberta Teta, and by the supervisor Prof. Alfonso Mangoni, and his work is being and will be continued by them. Dr. Francesca Rondinelli worked under the scientific supervision of Prof. Caterina Fattorusso. Her skills in quantum-mechanic calculations, using in particular the Density Functional Theory (DFT) applied to organometallics, perfectly filled the lack of expertise of our computational laboratory. She shared her expertise with the NeaNAT group, showing her results in a number of presentations to our computational group. The new expertise acquired by our research group from Dr. Rondinelli strongly improves the quality of the information obtained for the design on new anticancer agents. Indeed, all the molecular target taken in to consideration are enzyme catalyzing a metallo-mediated reaction. By consequence, the design of new druggable inhibitors of these enzymes (potential anticancer agents) needs computational techniques able to properly calculate the interaction with the metal as ab initio methods. Overall, the two years recruitment of Dr. Francesca Rondinelli as expert researcher in ab intio quantum mechanical methods within the NeaCADD group produced a requested and rather rare multi-disciplinary know-how which was permanently acquired by our research group. In the frame of Exchange of Knowledge activities, four bilateral agreements were signed with University of Bonn (group of professor Piel), University of Barcelona (groups of professor Lopez-Legentil and of professor Lupe), and with University of East Anglia (group of professor Goss). Dr. Roberta Teta spent 8 months at the University of Bonn and was trained in the most recent techniques used to relate the biosynthetic pathways of micro-organisms with the metabolites actually found in them. In particular, Dr. Teta was involved in the analysis of the genomes of some bacteria, the prediction of the metabolites that they may produce, and the search for the metabolites in extracts from the bacteria. The collaboration was very productive and a joint scientific paper of our and Professor Piel's groups has already been published, while other are in preparation. After her return, Dr. Teta is exploiting her new competence to continue this kind of work in our laboratory, both in collaboration with Piel's group and independently. The professional growth of Dr. Teta after her stay in Bonn is apparent. Not only did she learn several techniques that she can now use in our labs, but also improved her capacity of working independently and of designing and developing new projects. Dr. Bruno Catalanotti, permanent researcher at the NeaNAT group and leader of WP4 for Tasks 4.2 and 4.3 spent two months at the Departament de Fisicoquímica of University of Barcelona. Dr. Catalanotti received a further training in quantum mechanical calculations and was involved in the application of ab-initio calculation to the parametrization of small molecules. This secondment was very productive and led to two collaborative projects between NeaNAT and Prof. Luque's group. After his return, Dr. Catalanotti is exploiting his new competence in many drug design projects in which is involved, such us the above mentioned projects in collaboration with Prof. Luque, and in the development of novel FAAH and COX2 inhibitors as potential analgesics. Four researchers from twinned institutions spent 1-2 months in our group, introducing in our group useful techniques for molecular biology. ? Dr. Christian Gurgui from Professor Piel's group spent 1 months at our labs working on screening of biosynthetic genes on bacterial isolates from the sponge Plakortis simplex. In addition to this, Dr. Gurgui introduced in our lab a new technique, called yeast recombination, which allows one to reassemble large PKS genes which can only be isolated as fragments. ? Dr. Lucia Pita-Galan from the group of Professor Susanna López-Legentil at University of Barcelona spent 2 months at our labs on a project on microbial diversity in marine sponges. To this purpose, Dr. Pita introduced in our research group a recent technique, Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, that represents a useful additional expertise for our group. ? Dr. Max Crüsemann from Professor Piel's group (University of Bonn) spent 1 month at our labs working on the identification of minor analogues of the bioactive bacterial metabolite hormaomycin. Dr. Crüsemann was mainly involved in isolation of the hormaomycin analogues, and introduced in our group new extraction and separation techniques of bacterial metabolites which were not familiar to the group. In addition, Dr. Crüsemann's visit further strengthened the connections with Professor Piel's group. ? Dr. Joseph Zarins-Tutt from the group of Professor Rebecca Goss at University of East Anglia spent 2 months at our labs on a project directed to the isolation of marine actinomycetes (the most prolific bacterial source of new metabolites) from marine sediments. Dr. Zarins-Tutt performed the entire isolation process in our labs, starting from sediments collected along the coasts of Campania to obtain at least 33 pure stains of actinomycetes. While the main focus of our research remains on the bacterial symbionts of sponges, the newly acquired capacity of isolating actinomycetes may be a useful additional skill for our group. Overall, the four ingoing secondments provided to NeaNAT useful additional expertise on specific topics as well as new connections. We also gained expertise by inviting outstanding scientists of all the fields related to drug discovery from natural products to spend periods of 1-2 weeks as visiting professors in our labs. ? Professor Piel (University of Bonn, Germany) is one of the leaders worldwide as to the study of biosynthetic pathways of symbiotic micro-organisms, and the steady and productive collaboration with his group is one of the most important results of NatPharma project. Professor Jörn Piel (University of Bonn, Germany) spent 1 week in our group, giving two seminars, and during his visit we had occasion to check the status of the ongoing collaborative projects, and to plan for new ones. ? Professor Haynes (Hong Kong University of Science and Technology) is a medicinal chemist expert in antimalarial drugs, particularly those containing peroxide functions. Professor Haynes spent 2 weeks at our group giving a short course of three seminars The presence of Professor Haynes at the NeaNAT group was important to compare our and his ideas on the mechanism of action of plakortin, the antimalarial peroxide compound which is one of the most important objects of study in our group. ? Professor Nohubiro Fusetani (Fisheries and Oceans Hakodate, Japan) in November 2011. Professor Fusetani is one of the most outstanding scientists in the field of marine natural products, and is well-known for his skill in elucidating the structure of exceptionally complex compounds. Professor Fusetani spent 2 weeks at the NeaNAT group presenting a cycle of four seminars. We had occasion to discuss with him all our ongoing research projects, and received many useful suggestions. The project also envisaged a number of dissemination activities to increase the visibility and reputation of the NeaNAT group. A website about the research group was developed, and researchers from the group participated in scientific meetings, mostly international, giving presentations, chairing sessions, and establishing useful connections. The most important dissemination event was the international meeting "NatPharma: Nature Aided Drug Discovery (NADD)" organized by our group as a means to establish new connections with academia and companies, and an occasion for the NeaNAT group to advertise its new competence to the international community. This was held on 05?08.06.2011 in the prestigious Aula Magna Partenope at Conference Centre of the University ?Federico II?, in a historical building in one of the most beautiful areas of Naples. In spite of the present economic difficulties and of the absence of any tradition (and therefore of a core of loyal participants) of the meeting, the NatPharma meeting had 148 participants from universities and enterprises, coming from 9 different European countries, but also from USA, China, Canada, Japan, Brazil and Thailand. The meeting comprised four days: after the opening ceremony and two plenary lectures in the afternoon of 05.06.2011 we had four half-day sessions on 06?08.06.2011 on the topics 1. Bioactive Marine Natural Products, 2. Microbial Genetics, 3. From Mechanism of Action to Drug Design, 4. The Final Goal: ?Druggable Compounds?, covering all the aspects of the drug discovery from natural products. In addition, in the morning session of 06.06.2011 members of the NeaNAT group (including Dr. Hochmuth and Rondinelli, the two researchers recruited in the frame of the NatPharma project) gave 5 oral presentations to illustrate the main outcomes of the project. From the scientific point of view, the conference was very successful. The level of the scientific communication was high, and the feedback from participant was very positive. The conference allowed our research group to establish several new connections and ideas. The NatPharma meeting was also a way to increase our visibility on media. We also organized two round tables in Naples and Brussels about our research. A round table was organized at the Campania Regional Council on 22.06.2011 with the participation of the president of the Regional Council Paolo Romano and some representatives of pharma companies. During the round table, the coordinator Prof. Ernesto Fattorusso presented the research activities of NeaNAT group, including the NatPharma project; a discussion followed on the chances and problems with the interaction between academic research groups and enterprises in Campania and in Italy. In addition to this, after the meeting we were informally informed about the program "Open Innovation Drug Discovery" launched from Eli Lilly. The venue chosen and the presence of the president of the Regional Council guaranteed a wide media cov-erage of the event (see below). A second round table was organized in Brussels at the European Parliament on 10.11.2011. This round table was not specifically planned in the project, and was a follow-up of the NatPharma workshop. The participants on the round table were representatives of three pharma companies, researchers from other European groups that we have contacted in the frame of the NatPharma project, the vice-presidents of Italian Chemical Society and of its Campania section, respectively, and the scientific officer of NatPharma Project, Dr. Colombe Warin. The scientific topic was about the biotechnological production of natural products and the opportunity for future collaborative European projects between academia and industry. In addition, there was as a speech from Raffaele Liberali (Dir. "Energy" , DG Research & Innovation - European Commission), who illustrated the perspectives of European support to research and innovation after the. end of the 7th Framework Programme and, in particular, some aspects of the incoming Horizon 2020 programme. In addition to a wide coverage in Italian media, the round table was the occasion to consolidate our connections with the other participating European research groups. As result of the activity of the press agent we hired for the duration of the project, our dissemination events (including the NatPharma workshop) and our research in general had a much wider coverage by generalist media than ever in the past. ? On 13.04.2011 an article about the NeaNAT group and the antimalarial molecule plakortin appeared on the national newspaper Il Mattino. This was followed by a TV report on the group including an interview to the coordinator Prof. Ernesto Fattorusso. ? The NatPharma workshop (5-9.06.2011) was covered by traditional and web newspapers and by a TV report including interviews to some members of the group, broadcasted from several local TVs. As a follow-up, on 09.06.2011 Prof. Ernesto Fattorusso gave a radio interview on RAI, the Italian national broadcasting company. ? The round table at the Campania National Council on 22.06.2011 led to a TV report with in-terviews to several members of the group, broadcasted from a local TVs. In addition, many articles about the round table appeared on traditional and web newspapers, including the national newspaper Roma. ? The round table in Brussels attracted the largest media attention. On 19.11.2011 a full page of the economics newspaper Il Denaro, and part of the first page, was dedicated to the NeaNAT group, the NatPharma project, and the round table in Bruxelles; the round table was also reported on the national newspapers Il Mattino and other local newspapers. Using the material recorded in the dissemination events, a DVD was prepared for distribution to potential industrial partners interested in our work. Potential Impact: The final result expected from NatPharma was the growth of the research potential of the NeaNAT group. This goal has been achieved, and the most significant results obtained are: i) the research facilities, ii) the human potential, and iii) the connections with other research institutions and with the local productive infra-structures. The NeaNAT group now owns state-of-art equipment for molecular biology, which is a necessary requirement to exploit the new expertise acquired through the NatPharma project, and in addition it can attract researchers from other groups to visit us and start collaborative projects. The analytical capacities of the NeaNAT group are remarkably increased by the Orbitrap high-resolution mass spectrometer, which will be used by all the researchers of the NeaNAT group, and will allow the group to provide analytical services to private companies and environmental monitoring services to local, national and European public organizations. The NeaNAT group also gained a powerful hardware and software computational infrastructure, that will be the basis of the work of the group in the next years. The human potential resulted greatly improved by the recruitment of two researchers expert, respectively, in the identification and expression of biosynthetic genes and in quantum-mechanical calculations for interactions of drug candidates involving metals. The recruited researchers worked in close contact with staff members, leading to an effective training of the whole NeaNAT staff, so that the required knowledge is now permanently acquired by the group, even though external conditions made impossible the permanent recruitment of new researchers. The ability to study the biogenetic pathway of a natural product is essential to keep NeaNAT in an outstanding position in its field. Expertise in the expression of biosynthetic pathways can be used to create easily-cultivable bacterial strains producing useful natural products, strongly increasing the chance of success in the development of drugs from natural products, and leading to collaboration with pharmaceutical industries interested to exploit the potential of marine natural products. The application of computational methods in drug design is greatly improved by the possibility to integrate quantum mechanical simulations, making it possible to challenge wide-ranging issues in medicinal chemistry and providing new tools to improve the design process. Since a large number of molecular targets of pharmaceutical interest are enzymes catalyzing a metallo-mediated reaction, the acquired knowledge will allow us to renew the academic collaborations and industrial research agreements already gained during the project, as well as to establish new research platforms either with academic or industrial partners. The strengthening of NeaNAT in terms of human potential and of equipment have already shown some results. The group is part of the European-Chinese Research Staff Exchange Cluster for Marine Biotechnology (call FP7-PEOPLE-IRSES), financed by the EC (Grant agreement no. 246987) in August 2010, and two researcher from Chinese universities are currently working in our group. The group has also applied, in a consortium of 16 research organizations and enterprises, to the call FP7-KBBE-2012-6 with the integrating project "BlueGenics". This project fits perfectly the theme of NatPharma project. The new expertise in drug design of the group makes it easier to offer research services to pharmaceutical companies. The group has already obtained two consulting research agreements with Sigma-Tau Research Switzerland S.A. and expects to further increase this activity. Thanks to NatPharma, NeaNAT might also contribute to the regional development. through the offer of its know-how to public organizations. As a result of the improved analytical capabilities and the higher reputation obtained though NatPharma, the group has reached a four-year agreement (2011-2015) with the Campania region to monitor the possible presence of toxins produced by blooms of the alga Ostreopsis ovata. In addition, the Department of Natural Products, where the NeaNAT group is based, is negotiating an agreement with Public prosecutor's office in Napoli for analysis of seized samples of drugs. NeaNAT will also work to establish a closer connection with research organizations and companies based in the Campania region and involved in biotechnological research. In this respect, the Campania region launched for the realization of a network for biotechnology financed through structural funds FESR 2007-2013, and the NeaNAT group will apply to be one of the actors in this network. List of Websites: http://www.neanat.unina.it/index.php?item=98 Professor Ernesto Fattorusso Dipartimento di Chimica delle Sostanze Naturali Università di Napoli Federico II via Domenico Montesano 49 80131 Napoli - Itay