Irreversibly Silencing Oncogenic Master-regulator cMyc Using Library-derived Electrophilic Helical Peptides

A human gene that has been found to be crucial in the development of over 70% of all cancers is known as MYC. This gene codes for a protein (called cMyc) that is responsible for transforming normal cells to cancer cells and promoting cancer cell growth and tumour formation.

cMyc is a difficult protein to target with antibody-based drugs because it exists inside the nucleus of cancer cells where antibodies cannot go. cMyc is also a difficult protein to target with small molecule drugs that can enter the nucleus of cancer cells but cannot readily block the interactions cMyc makes across large surface areas with other proteins and DNA. No small molecules are known to block cMyc-protein interactions, and only a few small molecules of very low potency block Myc-DNA interactions.

Here we will develop and test a new approach to generating therapeutics that can, in the long term, overcome these problems by silencing cMyc and kill Myc-expressing cancer cells without killing normal human cells.

We propose to screen libraries of amino acid-containing peptides inside cells to find those with the best ability to selectively bind to cMyc. We have developed a method for tightening the binding of peptides to the target protein through adding a chemical 'electrophile' that sticks the peptide to the protein like a safety pin mechanism. Optimised peptides will bind selectively to cMyc and not come off. This approach can chemically silence cMyc and prevent it from promoting cancer. Our preliminary data demonstrates that this approach can work in that we have found that we can irreversibly bind peptides to cMyc and stop its binding to protein partners present inside cells, potentially silencing its functions and causing cancer cell death without killing normal cells.

The proposal seeks to build upon our strong proof-of-concept pilot data to optimise the peptide sequences for:

(a) selective binding to the protein cMyc,
(b) tight and irreversible binding to the protein cMyc,
(c) maximum uptake of the peptides into cancer cells and further into their nucleus,
(d) irreversible binding to cMyc inside the nucleus of cancer cells,
(e) inducing death of a range of cMyc-expressing human cancer cells without killing normal cells.

Success in these endeavours will be of exceptional interest to pharmaceutical companies and medical researchers who are becoming excited about cMyc as a new target for cancer. We present an entirely new and viable therapeutic approach to a very promising new cancer target. The technology developed will be potentially transferable to other protein-protein interactions (PPIs) implicated in disease pathways.

cMyc is a master regulator of cancer progression and linked to development of most human cancers. We will use electrophilic peptides derived from Max-based libraries, a known binding partner that forms a cMyc-Max heterodimeric coiled coil, to selectively and irreversibly target and functionally silence cMyc. Since cancer cells depend on cMyc the approach can induce death without killing normal human cells.

We will first use intracellular peptide-library screening to identify peptides highly selective for cMyc, thereby providing a platform to inhibit function. Expressing off-target proteins in the assay (e.g. Max/Mad/Mnt/Mga), will enhance selectivity AND free Max to heterodimerise with them, to both silence transcriptional activation AND enhance repression. Intracellular selection will guide affinity/selectivity while removing non-specific, unstable, insoluble, or aggregate prone library members, leaving excellent scaffolds for further therapeutic refinement.

We will further modify optimised cMyc-selective peptide sequences by conjugation to an electrophilic 'warhead', itself optimised for reactivity, geometry and location to ensure irreversible, but selective, cMyc binding. Selectivity is driven by a peptide-cMyc coiled coil PPI, while the electrophile acts like a secondary potency-conferring safety pin for irreversible sequestration of cMyc (via a unique cys), that is rendered functionally silent. We have demonstrated that the approach works for other proteins inside cells and now provide preliminary data showing feasibility for cMyc.

Peptides and electrophilic peptides with greatest promise will be tested using a range of biophysical, structural, chemical, and cell-based approaches to demonstrate efficacy in entering cancer cells (intrinsically, via mimetic refinement, CPP or lipidic appendage see CfS), ablating cMyc function, resisting metabolism and killing a range of cMyc-expressing cancer cells at concentrations that do not kill normal human cells.

In this proposal we seek to derive small potent inhibitors of the cancer master-control switch, cMyc, which is a key player in >70% of all cancers. The project will utilise rational design of an electrophilic warhead moiety, which will be conjugated to peptides identified via intracellular 'Protein-fragment Complementation Assay' (PCA) screened libraries. We will compare PCA-derived linear sequences with helix-constrained downsized counterparts. These initial peptides and mimetics will deliver the project to the preclinical stage by providing a scaffold from which new drugs will be developed. This is a standard approach to developing new treatments. The ultimate goal of our work is to generate compounds as a starting point for therapeutics against a number of cancers in which cMyc is a key determinant, which can then be taken forward to preclinical testing. There is clearly enormous potential benefit to the public sector health provision costs. This research offers the potential to derive such a treatment, which as well as benefiting the public sector, will potentially transform the approach used to derive new inhibitors, by providing a pre-defined intracellular screen for efficacy.

We also expect that our research will lead to considerable benefits for academics attempting to generate functional inhibitors of protein-protein interactions (PPIs) both for therapeutic and non-therapeutic goals. Furthermore, the acceleration of the drug design process gained by using PCA to generate novel helix-constrained peptides can serve as the starting point for therapeutics against other disease processes involving helical PPIs. This has considerable commercial benefit as these advances from the basic level of understanding are exploited at the commercial level, carrying with it considerable benefits to the knowledge economy of the UK. The proposal will support two PDRAs (Bath/Queensland), who will be trained in the techniques outlined above and in addition will learn transferable skills such as conference networking, giving seminars, and writing reports, as well as an exchange programme to allow them to spend time at either institute. They will also benefit from the diverse experience gained by the close collaboration between the Mason and Fairlie groups, who will stay in regular contact via email, phone and video conferencing, as well as face-to-face meetings. Mason and Fairlie's initial collaboration was supported by a EPSRC travel grant (EP/M001873/2) leading to a number of additional major collaborations (BBSRC, BB/R017956/1; CRUK, A-26941; ARUK; ARUK-PG2018A-003) and publications centred around helix-based PPI disease targets (e.g. Rao, T., et al, PLOS One 2013; Mason & Fairlie Future Med. Chem 2015; Shepherd N.E., et al., Org Biomol Chem 2016; Baxter D., et al, ACS Chem Biol 2017; Meade R., et al., Mol Neurodegen 2019), with several more in preparation.

All findings in such a topical field will be publishable in high impact journals to disseminate with the wider scientific community. In addition, seminars will be hosted for scientists (invited seminars and meetings), the general public (e.g. cafe scientifique, school days at Bath, Bath Taps into Science events). Communications outside the university will be aided by the Communications Office at Bath who will work alongside the press office at Queensland to liaise with journalists for radio/television and other media pieces, and who also produce their own publication 'BA2'. A dedicated website will report on project developments and be fully accessible to readers of all levels. Relevant media pieces including podcasts and vodcasts will be placed onto the site.