Projects / Programmes
New technology for design of novel polyketide drug-leads with chemically ameanable moieties
| Code |
Science |
Field |
Subfield |
| 4.06.01 |
Biotechnical sciences |
Biotechnology |
Recombinant DNA technology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
Polyketides, polyketid synthases, acyltransferase, biosynthetic engineering, drug discovery
Researchers (15)
Organisations (2)
Abstract
Polyketides are a large group of biogenetically related natural products, synthesized by a diverse group of biosynthetic enzymes, termed polyketide synthases (PKS). They display astonishing structural diversity, reflected in a large spectrum of pharmacological activities of great clinical and industrial importance. Microbial resistance, non-optimal pharmacological properties and off-target activities of current drugs present a clear need for development of novel compounds of this class. Semi-synthetic derivatisation has resulted in many clinically successful molecules. However, polyketides offer only a limited number of chemically amenable functional groups, thus significantly limiting development of new semi-synthetic routes. Further possibilities emerged with biosynthetic engineering of PKS enzymes, particularly those classified as type I. These enzymes are organized in modules, containing sets of dedicated enzymatic domains in a manner that each module catalyzes one step of the polyketide chain extension, analogously to fatty acid biosynthesis. Targeted modifications of polyketide structures can be obtained by modifying the domains in a particular module. Among such modifications, replacement of acyl transferase (AT) domains, which determine the choice of each extender unit during the polyketide biosynthesis, has been often used. Almost exclusively, extender units used in polyketide biosynthesis are limited to malonyl-, methylmalonyl- and ethylmalonyl-CoA, thus allowing only a limited structural diversity of polyketide compounds. In fact, these limitations have not been overcome in biosynthetic engineering as almost all successful AT replacement experiments resulted only in removal or addition of methyl or ethyl groups to the existing polyketide backbones. Such derivatives have generally not shown improved pharmacological properties, and due to chemical inertness of methyl group, it was not possible to expand structural diversity through semi-synthetic derivatisation. We have recently discovered that an unusual AT module 4 from PKS of the immunosuppressant FK506 (fkAT4) can incorporate unnatural building blocks with chemically reactive terminal double and triple bonds as well as other unnatural extender units (unpublished data). Even more importantly, this AT domain does not incorporate universally abundant malonyl-CoA and methylmalonyl-CoA, thus presenting a unique potential for biosynthetic engineering of polyketides. We therefore propose development of a new technology platform, which would enable introduction of chemically amenable side chains into any Type I polyketide-derived metabolite, thus generating enormous opportunities for semi-synthetic derivatization and thereby unprecedented chemical diversity of potential novel compounds. 3D-modelling approach will first be applied to gain additional insight into specificity of the unusual fkAT domain and assist in optimum junction site selection for construction of hybrid PKS genes. This will be followed by “In vitro” biochemical analysis of purified proteins containing the fkAT4 domain together with other domains of its native module using different synthetic extender unit derivatives. Simultaneously, we will also carry out “in vivo” analysis of the functionality of novel hybrid PKS genes. These will be done on the basis of the erythromycin PKS and DEBS1-TE model systems with an inserted fkAT4 domain. Using a chemobiosynthetic approach, novel analogs will be generated by the hybrid PKS genes. Site-directed and random mutagenesis will be used to generate novel variants of the fkAT4 domain with further optimized specificity profile. Thus, in the scope of this project proposal, we will aim to demonstrate the potential applicability of the proposed technology platform, which could be used for derivatization of numerous polyketide drugs of medical importance by generating novel analogues with chemically amenable moieties.
Significance for science
Biosynthetic engineering of PKS enzymes and subsequent semi-synthetic derivatization of produced compounds ensured development of a number of novel drugs. However, a better understanding of the PKS biosynthetic activity, particularly of the specificity/selectivity of AT domains, still remained essential for a more efficient generation of novel compounds and drug leads with improved pharmacological properties. We were focused on improving our understanding of PKS specificity and biosynthetic mechanisms involved in the production of these medically important metabolites. Particularly, this study was focus on the specificity of acyltransferases (ATs), which are the key domain of every PKS module involved in the selection of extender units, thus significantly influencing the chemical structure of polyketide-based drug. Very limited diversity of extender units used by PKS enzyme, and especially their chemical "inertness" for semi-synthetic chemistry approaches is the major disadvantage of biosynthetic machinery of PKS. Therefore, it was one of the main aims of this project to expend the "arsenal" of extender units, which will offer novel semi-synthetic modifications. Although intensive efforts have been invested into understanding of AT selectivity in the past by leading institutions globally, this scientific area still presents many important unanswered questions. During the progress of this project, some of the competing institutions and research groups in Germany, in the US and in South Korea, were attempting to solve the problem of the diversity/selectivity of building units used by PKS enzymes, but were largely unsuccessful. In the scope of this project, which considerably differs from other approaches, we have made significant progress on the platform with strain S. tsukubaensis. We have successfully biosynthesised the first compound which now offers diverse approaches of semi-synthetic chemistry. We have intensively studied, and identified a novel and very unique biosynthetic pathway involved in the formation of the unusual allylmalonyl-CoA extender unit, which is selected and incorporated by the unique AT4 domain of the FK506 PKS cluster (Goranovic et al. 2010, Kosec et al., 2012, US 8980585 (B2)). Following these initial findings, at Acies Bio we have realised the great potential of the allylmalonyl-CoA biosynthetic machinery and the uniqueness of the AT domain of FK506 module 4 (fkAT4 domain). Experiments carried out in a heterologous host system S. erythraea unfortunately did not give satisfying results, similarly to results from German research group (Klopries et al., 2015). Our experimental data are suggesting that AT domain is not the only one affecting the selectivity and specificity of this complex system (Blažič s sod 2015). Extension of polyketide chain on the module 4 FK506 PKS is probably not mediated by CoA-activated extender units but rather directly in ACP-linked form. Our research data further suggests that we should transfer complete enzymatic system for the biosynthesis of this unusual extender unit from FK506 into heterologous industrial host strain of S. erythraea (Karničar s sod. 2016), which is not a simple task. In contrast,, the chemo-biosynthetic approach, and platform developed in S. tsukubaensis proved to be very productive. We were probably the first research group to prepare target polyketide with a triple bond in sufficient quantities using chemo-biosynthetic approach, which can be further transferred to the industrial scale. Thereof, the understanding of the background of biosynthesis of unusual extender units in S. tsukubaensis was crucial (Blažič et al., 2015). During the period of this project, we managed to evaluate and optimize the new platform and successfully produce novel drug candidate base on immunosuppression drug FK506, which shows very interesting biological activity, and thus significantly deepened overall our understanding on the activity of this complex enzymatic system.
Significance for the country
Extensive efforts have been invested into the studies on polyketide biosynthesis, which is not surprising considering the great medicinal and industrial importance of numerous polyketide- derived active compounds, which currently represent a market of over 50 billion USD. Polyketides are being intensively used in medicine and agriculture due to their remarkably wide array of biological activities. There is still a great demand for development of effective therapies, especially in the fields of anti-infectives, immunomodulation and oncology and a large number of polyketide drug candidates are currently in clinical trials. In Slovenia, this field is of great importance as both Slovenian pharmaceutical companies, Lek/Sandoz and Krka, produce numerous drugs based on these compounds. This field is also important for Acies Bio who is developing both genetic as well as innovative technologies for production of these pharmaceutical active ingredients in the form of contract research projects for large national and global pharmaceutical and biotechnological companies or in the form of in-house research projects, developing novel bioprocesses and novel biologically active ingredients. Currently, Acies Bio is actively working on two groups of biologically active compounds, involving treatment of bacterial infections, rare neurodegenerative diseases and anticancer drugs. In the scope of this three-year project Acies Bio developed a new platform, which will enable the development of completely new matrices, which could not be obtained using existing technologies. One of the FK506-based leads, which we produced, is showing interesting biological activity, but reduced toxicity compared to the starting compound. Testing of this new compound is ongoing in collaboration with the Helmholtz institute in Germany and is expected to be concluded by July 2017. The proposed technological platform presents a new opportunity for development of new structural matrices based on existing clinically proven successful polyketides, polyketide leads currently in clinical trials and promising compounds that didn’t qualify for clinical trials due to unwanted adverse effects. This technology can be used for development of important new drugs or drug leads with significantly improved pharmaceutical and therapeutic properties, which could benefit the wider society as well as increased competitiveness of the Slovenian biotechnology and pharmaceutical industry in general. The new technology platform enables important technological progress and raises competitiveness of biotechnological company Acies Bio who is co-financing the project. Execution of the project included international efforts by the research group formed by biologists, microbiologists, synthetic chemists and structural biologists. This consortium will continue cooperating, considering it presents an excellent platform for the transfer of knowhow and technology between academia and industry. In the financial scope of the proposed research project, we also trained young researchers and post-doctoral researchers, which were collaborating with the dynamic biotechnology company Acies Bio. The new knowledge, expertise and competitive advantage, which have been acquired during the course of this, still ongoing project, will offer support for further development of the Slovenian society. Based on the results of the executed project, we are expecting a new publication in an international journal with a high impact factor and presentation of our work to the wider scientific society at conferences, which will additionally contribute to the promotion of Slovenian science in the field of novel drug development.
Most important scientific results
Annual report
2013,
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2013,
2014,
2015,
final report
