Synthesis of glycosyl-novobiocins: probes of Hsp90 C-terminal affinity binding and novel anti-cancer drugs

Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths) in 2008 (WHO cancer, 2012). There are an estimated 12.7 million cancer cases around the world every year, and this number expected to increase to 26 million by 2030.Chemotherapy has proven to be very useful in addition to surgery in treating cancer. However, its effectiveness is often limited with the number of useful drugs and their toxicities. Designing effective drugs are always required to treat cancer for public health. Here we report a new approach to target Heat Shock Protein 90 kDa (Hsp90) using glycosylation technology. Hsp90 mediates protein folding which is important to cancer cell survival. Inhibition to Hsp90 maysimultaneously inhibit multiple therapeutic targets and pathways crucial to tumour survival.
Novobiocin was originally approved for clinical use in the 1960s under the trade name Albamycin (Pharmacia and Upjohn) as an antibiotic drug. Recently, Novobiocin has also been shown to have low anticancer activity via binding to Hsp90. Previously, the poor affinity to Hsp90 and higher affinity for type-II topoisomerases prevented novobiocin being evaluated as a clinical useful Hsp90 inhibitor. Recently, we demonstrated that a glycosylation approach can separate the anti-cancer activity from the antibacterial activity (as a proxy of topoisomerase) to 27,000 fold providing a useful alternative strategy for anti-cancer drug discovery. However, the mechanism on how the drug interacts with Hsp90 C-terminal is not fully clear.
The important biological roles of glycosylated novobiocins suggest that carbohydrate modifications are central to the biological function of these molecules. We propose that these glycosyl-based modification strategies are a potential route to the rational design, modification of lead compounds and repurposing of existing drug molecules.

This project will provide full understanding of the Hsp90 Structure-Activity Relationship which will benefit academic research in this area with detailed information and guidance to develop inhibitors that can be lead to clinical trial.
In addition, the newly developed C-terminal binding assay will provide a robust and convenient method to determine and quantify the inhibition type (C or N) and the effect (IC50). The Hsp90 ATPase assay will also provide information on inhibition pattern and constant.
The results from this research will not only add to our basic scientific knowledge in this area benefitting researchers, teachers and students in academia and scientists in the commercial sector, but it will also lead to potential new drugs. Furthermore, glycosylated novobiocin analogues could potentially be developed as therapeutic agents that will bring benefit to the wider public.
2. Main Areas of Impact
(a) University Sector: Modifications on novobiocin to achieve better activity have been undertaken by various approaches. However, this is the first time that glycosylation technology has been used and results in separating antibacterial activity and enhancement of anticancer activity are highly encouraging.
The mechanism of how the novobiocin or its glycosylated analogues interact with Hsp90 is still not fully understood. In this project, we plan to synthesize photoaffinity labelling reagents so that we can probe the SAR of novobiocin analogues around Hsp90. This will provide detailed information on how novobiocin and especially the sugar moiety are interacting in the binding mechanism. This will give guidance and inform our synthesis of analogues with improved activity.
The techniques used in this project will facilitate the ability of researchers working in this area. For example we have developed a number of useful techniques to study these interactions and these include Hsp90 binding assay, an ATPase assay, an MS uptake assay, protein modelling, the use of a Western Blot assay and finally anticancer (MTT) and antibacterial assays. The selective glycosylation approach will clearly benefit researchers working in carbohydrate synthesis.
By appropriate dissemination of results from this research programme (e.g., scientific journals, general-interest publications, university seminars, conference lectures and posters, and web blogs), it is anticipated that information relating to the novel anticancer and antibacterial drugs will reach lecturers in universities, teachers in schools and tertiary colleges. Finally, clinical academic researchers should be interested in compounds from this research to develop novel therapeutic agents (e.g., novel antibacterials and oncology agents).
(b) Commercial Sector: Novobiocin was licenced for clinical use under the tradename Albamycin (Pharmacia and Upjohn) in the 1960s. Novobiocin is an effective anti-staphylococcal agent used in the treatment of MRSA. It has however been withdrawn from the market due to its low solubility, low activity and toxicity. Novel glycosylation technology has re-invigorated interest in novobiocin as a broad-spectrum anticancer drug. This will have direct benefit to scientists in parts of the commercial sector. Results of our research have been filed in a patent application through the School of Pharmacy's Commercial Development Office prior to publication.
(c) Wider Public: Members of the wider public are most likely to benefit from this research project through the possible introduction of novel therapeutic anticancer drugs and antibiotics. Glycosylated novobiocin has shown good broad spectrum anticancer activity against breast, lung, brain and pancreatic cancer cell lines and moderate activity in an ovarian cancer cell line. Development of analogues of novobiocin also have great potential given the bactericidal nature of novobiocin.