Development of a multifunctional benzophenone linker for peptide stapling and photoaffinity labeling

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker, which serves as both a photocrosslinking motif and a peptide stapling reagent. Using a double-click stapling methodology, we attach the benzophenone to the peptide via the staple linker, rather than modifying the peptide sequence with a photocrosslinking amino acid. Applied to a p53-derived peptide, the resulting photoreactive stapled peptide is able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.This work was supported by the EPSRC, BBSRC, MRC, Wellcome Trust and ERC (FP7/2007-2013; 279337/DOS). We thank Dr. Clemens Mayer for access to the UV crosslinker (University Chemical Laboratory, University of Cambridge), Weiyan Chen and Fran Kundel (University Chemical Laboratory, University of Cambridge) for assistance with the Typhoon imager and Dr. Laura Itzhaki and Wenshu Xu (Department of Pharmacology, University of Cambridge) for assistance with SDS-PAGE.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/cbic.20150064

Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling.

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.This work was supported by the EPSRC, BBSRC, MRC, Wellcome Trust and ERC (FP7/2007-2013; 279337/DOS). We thank Dr. Clemens Mayer for access to the UV crosslinker (University Chemical Laboratory, University of Cambridge), Weiyan Chen and Fran Kundel (University Chemical Laboratory, University of Cambridge) for assistance with the Typhoon imager and Dr. Laura Itzhaki and Wenshu Xu (Department of Pharmacology, University of Cambridge) for assistance with SDS-PAGE.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/cbic.20150064

The Identification and Development of Small Molecule Inhibitors of Amyloid β Aggregation

Amyloid $\beta$ (1-42) (A$\beta$42) is a seminal neuropathic agent in Alzheimer’s disease (AD), a multifaceted neurodegenerative disorder for which no preventative measures or disease modifying therapies currently exist. Aggregation of this peptide plays a key role in the synaptic dysfunction and neuronal death associated with the disease. Perturbing the aggregation process, therefore, represents a key strategy for the development of new AD therapeutics. A variety of issues with current screening methods, including lack of reproducibility, high reagent consumption and spectral interference from the test molecules, can limit efforts to identify new small molecule inhibitors. Furthermore, the lack of robust, time- and cost-efficient methods for screening compounds in cellular or in vivo models limits the throughput with which seemingly active small molecules can be validated and prioritised. Herein, this thesis describes efforts to overcome such limitations through the development of a unified in vitro to in vivo assay system, in which hits identified in the ‘nanoFLIM’ microfluidic-based assay can quickly be tested in cellular and whole organism disease models. The assay platform designed relies on the use of an amyloid aggregation fluorescence lifetime sensor. A$\beta$42 aggregation is monitored by changes in the fluorescence lifetime of an attached fluorophore, which is significantly quenched upon amyloid formation. To take advantage of the benefits associated with miniaturisation, an in vitro microfluidic platform was employed. A microfluidic chip capable of trapping 110 precisely ordered droplets was designed, allowing for increased sample size and greatly lowering reagent consumption relative to conventional assay formats. Optimisation of the lifetime sensor technique permitted real-time compound screening in SH-SY5Y neuroblastoma cells, as well as in disease model Caenorhabditis elegans (C. elegans). To demonstrate the potential of this assay, a selection of novel chemical libraries developed in the Spring research group was screened, resulting in the identification of a key library of interest. The inhibitory activity of the lead compound from this collection was validated using a variety of biophysical tests, and was also shown to suppress amyloid aggregation in the live cell fluorescence lifetime sensor assay, as well as in whole organism disease model C. elegans. Whilst assay development was underway, additional screening of structurally diverse chemical libraries was performed using a conventional Thioflavin T spectroscopic assay. Such work identified another molecular scaffold capable of exerting a strong inhibitory effect against A$\beta$42 aggregation. A selection of analogues was synthesised to improve the in vivo profile of this library, giving rise to a second lead inhibitory compound. The activity of this compound was subsequently validated in biophysical and cellular tests, and was also tested in disease model Drosophila melanogaster. The aggregation of A$\beta$42 lies at the root of Alzheimer’s disease. In light of the relatively few drug candidates in clinical trials for this disorder, the development of improved translational screening approaches and continued screening of novel chemical libraries is necessary to identify new potential therapeutics. In this study, an in vitro to in vivo fluorescence lifetime imaging assay has been established. Using this assay system and conventional screening approaches, two A$\beta$42 aggregation inhibitors have been identified and validated. These represent promising candidates for the development of new AD therapeutic agents, or for use as molecular probes to further dissect the mechanisms underlying this devastating disease.Biotechnology and Biological Sciences Research Council (BBSRC

Divergent synthesis of biflavonoids yields novel inhibitors of the aggregation of amyloid β (1-42).

Biflavonoids are associated with a variety of biologically useful properties. However, synthetic biflavonoids are poorly explored within drug discovery. There is considerable structural diversity possible within this compound class and large regions of potentially biologically relevant biflavonoid chemical space remain untapped or underexplored. Herein, we report the development of a modular and divergent strategy towards biflavonoid derivatives which enabled the step-economical preparation of a structurally diverse collection of novel unnatural biflavonoids. Preliminary studies established that the strategy could also be successfully extended to the preparation of very rare triflavonoids, which are also expected to be useful tools for biological intervention. Prompted by previous inhibitory studies with flavonoid libraries, amyloid anti-aggregation screening was performed, which led to the identification of several structurally novel inhibitors of the aggregation of the amyloid β peptide (Aβ42). Aggregated Aβ42 is a pathological hallmark of Alzheimer's disease and the use of small molecules to inhibit the aggregation process has been identified as a potentially valuable therapeutic strategy for disease treatment. Methylated biaurones were associated with highest levels of potency (the most active compound had an IC50 value of 16 μM), establishing this scaffold as a starting point for inhibitor development.We thank the Cambridge Commonwealth Trust and Cambridge Home and European Scholarship Scheme for the awards of scholarships to T. H. S., T. J. S and S. C. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreements no. [279337/DOS and 695669 & 665631/FH]. The authors also thank AstraZeneca, the European Union (EU), the Engineering and Physical Sciences Research Council (EPSRC), the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), and the Wellcome Trust for funding

Synthesis of a novel polycyclic ring scaffold with antimitotic properties via a selective domino Heck-Suzuki reaction.

The synthesis of a previously undescribed sp3-rich 6-5-5-6 tetracyclic ring scaffold using a palladium catalysed domino Heck-Suzuki reaction is reported. This reaction is high-yielding, selective for the domino process over the direct Suzuki reaction and tolerant towards a variety of boronic acids. The novel scaffold can also be accessed via domino Heck-Stille and radical cyclisations. Compounds based around this scaffold were found to be effective antimitotic agents in a human cancer cell line. Detailed phenotypic profiling showed that the compounds affected the congression of chromosomes to give mitotic arrest and apoptotic cell death. Thus, a novel structural class of antimitotic agents that does not disrupt the tubulin network has been identified