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Anchoring ligandable binding sites at E3 ligase surfaces for plug-and-play PROTACs.

Periodic Reporting for period 1 - ANCHOR E3s (Anchoring ligandable binding sites at E3 ligase surfaces for plug-and-play PROTACs.)

Reporting period: 2016-08-01 to 2018-07-31

Many human diseases, including cancer, are often characterised by the malfunctioning or misregulation of specific proteins. A general therapeutic approach to improve the health of diagnosed patients is to use chemical compounds that bind to the active site of the protein and inhibit its action. In our research group we tackle the problem from a different perspective by developing so-called PROteolysis-TArgeting Chimera (PROTAC) compounds. These molecules bind a protein of interest that we aim to destroy and an E3 ubiquitin ligase, which targets proteins for degradation within the body. To summarise, PROTACs hijack the natural functioning of the cellular machinery to trigger selective degradation of the protein of interest. Since PROTACs do not require binding to a functional site of the protein, there is mounting expectation regarding their use to treat currently challenging pharmacological disorders, such as specific cancer types.
The current project focusses on developing computational tools to discover novel binding sites in proteins, and to use molecular modelling tools to study different aspects of PROTAC design and mode of action. This is important to ensure that the technology reaches the desired levels of usability and to expand its applicability scope. Notably, the first targeted protein degraders are expected to enter oncology clinical phase I studies by the end of 2018.
I initially developed a large library of small molecules extracted from catalogues of commercially available compounds. Our library design is unique because it is developed with a focus on proteolysis-targeting chimeras. Starting from over 74 million compounds, I prepared a carefully chosen, diverse selection of ~500,000 compounds for computational screening, and we also set up a local library of ~1,000 molecules for use within our research group and the Drug Discovery Unit in Dundee. These fragment libraries were extensively used within and beyond the project. I first screened for compounds binding to the PHD domain of BAZ2A and BAZ2B proteins and identified the first small molecules binding to these domains (Amato et al., ACS Chem. Biol., 2018). Additionally, I carried out modelling studies to unravel the recognition mechanism of the histone tail, where DNA wraps within the nuclei, by the PHD domains at the molecular level (Bortoluzzi et al., Biochem. J., 2017).
Another aim of my project was to implement and apply computational tools to identify binding sites in protein surfaces, and I applied them to discover binding sites in the surface of VHL, an E3 ubiquitin ligase involved in the physiological response to low-oxygen conditions and a common target hijacked by proteolysis-targeting chimeras (Lucas, van Molle, and Ciulli, J. Med. Chem., 2018). The structure-based optimisation of fragments binding to these cavities are an ongoing project within the research group. During the development of the pocket discovery campaign, I also found inspiring evidence of an additional binding site in the E3 ligase. Therefore, I embarked on a structure-based endeavour to design, synthesise, and test cyclic peptides that could interact with this region of the protein. This represented a unique opportunity from a career development perspective, since it provided hands-on experience in many experimental techniques beyond the computational field.
I also performed molecular modelling studies to deepen our understanding of PROTAC design and mode of action. We perturbed binding of a VHL-hijacking PROTAC by substituting specific atoms in its VHL-binding warhead. A first investigation following this strategy revealed the crucial role of a specific amino acid in VHL in ligand recognition (Soares et al., Bioorg. Med. Chem, 2018). Second, we added a fluorine atom to the VHL ligand, and investigated its impact in the behaviour of the small molecule. This is revolutionary, because the project involved creating a novel artificial amino acid that has potential applications in several research areas including protein engineering. In this project, I provided molecular modelling expertise that enabled interpretation of experimental data. Conversion of this fluorinated VHL ligand into a PROTAC generated a very potent degrader of a protein of interest (Testa et al., J. Am. Chem. Soc., 2018).
Finally, during my Action our group also obtained the first crystal structure of a PROTAC bound simultaneously to its two target proteins: the protein of interest and the E3 ubiquitin ligase. I performed extensive molecular dynamics simulations to study and manipulate the behaviour of the ternary complex in solution (partly published in Gadd, Testa, Lucas et al., Nat. Chem. Biol., 2017). The crystal structure further enabled the structure-based design of more selective PROTACs, which will be published soon. I further co-authored a review on E3 ubiquitin ligases and selectivity of substrate recognition (Lucas and Ciulli, Curr. Opin. Struct. Biol., 2017).
The presented action has reached unforeseen results and has expanded well beyond the initially declared aims. The research opens opportunities in the chemical biology and drug discovery fields, and has already shaped the academic and industrial study of molecule degraders as therapeutic agents. Results of the research have appeared in several scholarly publications and have been presented in posters and talks in international scientific conferences.
As part of the project, I have developed a fragment library that has been extensively used as part of the project and has provided chemical matter to complementary projects within the University. My computer-based binding site discovery campaign has revealed several binding sites in the VHL E3 ubiquitin ligase, and can be analogously applied to the discovery of binding sites in other E3 ubiquitin ligases for which no small-molecule binder currently exists. This is crucial, because proteolysis-targeting chimeras require that a binder of an E3 ligase is readily available for conjugation, and justifies the work done during this Action. Importantly, I have shown that the developed fragment library can be used to identify binders of challenging proteins for which no binder is known. Taken together, the presented tools and resources have direct application in PROTAC development beyond the specific selected case-studies.
The publication of the first crystal structure of a PROTAC in ternary complex with an E3 ligase and a protein of interest has opened enormous opportunities for structure-based design of molecule degraders, and this is only starting to become apparent. Indeed, the publication has reached over 50 citations in the first year since publication and marks an important milestone in the field. Additionally, we have very recently published a novel artificial amino acid with direct application in targeted protein degradation by PROTACs, as well as unforeseen academic and industrial applications in other fields such as protein engineering.
Mechanisms of natural and induced targeted protein degradation.