Glycosylation and Glycodiversification in Polyketide Antibiotics: Unraveling Biosynthetic Steps in Nogalamycin Formation

Soil-dwelling Streptomyces bacteria are known for their ability to produce biologically active compounds such as antimicrobial, immunosuppressant, antifungal and anticancer drugs. S. nogalater is the producer of nogalamycin, a potential anticancer drug exhibiting high cytotoxicity and activity against human topoisomerases I and II. Nogalamycin is an anthracycline polyketide comprising a four-ring aromatic backbone,a neutral deoxy sugar at C7, and an amino sugar attached via an O–C bond at C1 and a C–C bond between C2 and C5´´. This kind of attachment of the amino sugar is unusual thus making the structure of the compound highly interesting. The sugar is also associated with the biological activity of nogalamycin, as it facilitates binding to DNA. Furthermore, the sugar moieties of anthracyclines are often crucial for their biological activity. Together the interesting attachment of the amino sugar and the general reliance of polyketides on the sugar moieties for bioactivity have made the study of the biosynthesis of nogalamycin attractive. The sugar moieties are typically attached by glycosyltransferases, which use two substrates: the donor and the acceptor. The literature review of the thesis is focused on the glycosylation of polyketides and the possibilities to alter their glycosylation patterns. My own thesis work revolves around the biosynthesis of nogalamycin. We have elucidated the individual steps that lead to its rather unique structure. We reconstructed the whole biosynthetic pathway in the heterologous host S. albus using a cosmid and a plasmid. In the process, we were able to isolate new compounds when the cosmid, which contains the majority of the nogalamycin gene cluster, was expressed alone in the heterologous host. The new compounds included true intermediates of the pathway as well as metabolites, which were most likely altered by the endogenous enzymes of the host. The biological activity of the most interesting new products was tested against human topoisomerases I and II, and they were found to exhibit such activities. The heterologous expression system facilitated the generation of mutants with inactivated biosynthetic genes. In that process, we were able to identify the functions of the glycosyltransferases SnogE and SnogD, solve the structure of SnogD, discover a novel C1-hydroxylase system comprising SnoaW and SnoaL2, and establish that the two homologous non-heme α-ketoglutarate and Fe2+ dependent enzymes SnoK and SnoN catalyze atypical reactions on the pathway. We demonstrated that SnoK was responsible for the formation of the additional C–C bond, whereas SnoN is an epimerase. A combination of in vivo and in vitro techniques was utilized to unravel the details of these enzymes. Protein crystallography gave us an important means to understand the mechanisms. Furthermore, the solved structures serve as platforms for future rational design of the enzymes.

Compulsory licensing of standard-essential patents and injunctive relief as abuse of dominant position in violation of Article 102 TFEU

When a dominant undertaking holding a standard-essential patent uses its exclusive right to the IP to seek injunctions against those wishing to produce either de jure or de facto standard compliant products, it creates a conflict between the exclusive right to the use of the IP on the one hand and the possible abuse of dominance due to the exclusionary conduct on the other. The aim of the thesis is to focus on the issues concerning abuse of dominance in violation of Article 102 TFEU when the holder of the standard-essential patent seeks an injunction against a would-be licensee. The thesis is mainly based on the most recent ECJ case law in Huawei and the Commission’s recent decisions in Samsung and Motorola. The case law in Europe prior to those decisions was mainly focused on the German case law from Orange Book Standard which provided IP holders great leverage due to the almost automatic granting of injunctions against infringers. The ECJ in Huawei set out the requirements for when a de jure standard-essential patent holder would not be violating Article 102 TFEU when seeking an injunction, requiring that negotiations in good faith must take place prior to the seeking of the injunction and that all offers must comply with FRAND terms, thus limiting the scope of case law derived from Orange Book Standard in Germany. The ECJ chose not to follow all of the reasoning the Commission had laid out in Samsung and Motorola which provided a more licensee-friendly approach on the matter, but rather chose a compromise between the IP holder friendly German case law and the Commission’s decisions. However, the ECJ did not disclose how FRAND terms themselves should be interpreted, but rather left it for the national courts to decide. Furthermore, the thesis strongly argues that Huawei did not change the fact that only vertically integrated IP holders who have made a FRAND declaration are subject to the terms laid out in Huawei, thus leaving non-practicing entities such as patent trolls and entities that have not made a FRAND declaration outside its scope. The resulting conclusion from the thesis is that while the ECJ in Huawei presented new exceptional circumstances for when an IP holder could be abusing its dominant position when it seeks an injunction, it still left many more questions answered, such as the meaning of FRAND and whether deception in giving a FRAND declaration is prohibited under Article 102 TFEU or not.