A Protein Functionalization Platform Based on Selective Modification at Methionine Residues

Nature routinely carries out site-selective modification of proteins, enabling a dramatic increase in functional diversity. In contrast, synthetic manipulation of proteins is restricted by the availability of suitable chemical transformations. However, access to synthetically modified proteins has become fundamentally important to chemical biology, molecular biology & medicine. This has stimulated intensive research into the development of chemical transformations that are compatible with biological systems. Ideally, a reaction should be selective at a single site on a protein at a rate that is commensurate with the kinetic demands of complex molecules; should operate under ambient conditions to prevent disruption of the protein architecture or function; & provide homogeneous products in near perfect conversion. Despite these challenges, the past 20 years have seen a number of exciting methodologies emerge for executing transformations, both in vitro & in vivo, at natural & non-natural amino-acid residues in proteins. While most chemical methods have focussed on expanding the toolkit for reaction at cysteine (Cys) & lysine (Lys) residues, there has been burgeoning interest in transformations at non-natural amino acids (via genetic encoding) that display side chain functionality with orthogonal reactivity to standard residues. Reagents that probe biological processes tend to rely on relatively simple reactions to circumvent problems with chemistry in complex environments. However, there is a need for complementary tools (to reactions at Cys & Lys) that selectively produce functional protein conjugates via previously unexplored amino acids.

Methionine (Met) is a proteogenic amino acids & displays a number of features which make it potentially amenable as a bioconjugation target: For example, Met has a <2% abundance in proteins, is easily encoded, has a limited role in ancillary protein function (mainly protection against oxidative stress) & contains a possible reactive handle via its weakly nucleophilic S-atom. Until recently, practical Met-selective bioconjugation was unknown. Concurrent with our initial work, Chang et al reported a redox-activated tagging strategy that converted Met to sulfoximine-derivatives & successfully applying it in biology-driven applications. We have developed a methionine-selective protein functionalization strategy based on the use of hypervalent iodine reagents (we call these reagents MetSIS, meaning Methionine Selective Iodonium Salts) that react selectively with polypeptides & proteins, often giving >95% conversion to a stable diazo-sulfonium conjugate at low concentration in H2O in <1 minute. Our initial work on Met-bioconjugation was recently published Nature 2018, 562, 563-568.

The structural diversity of the proteome in any single organism means that no one protein functionalization method will provide universal solutions to the preparation of protein constructs. Cys-ligation is the benchmark for protein labelling and is is fast, selective and continues to evolve powerful methods that can be applied to a plethora of chemical biology & bio-medical applications. Even though Cys has founded many distinct applications, what if another amino acid can be generally harnessed for protein functionalization that is complementary to Cys? Here, I propose that chemical targeting of methionine (Met) can be the basis of distinct strategies for protein modification. Through this Fellowship, I outline a program for synthesis-driven protein functionalization, where these biomacromolecules are labelled at Met for use in vitro & (possibly) in vivo environments thereby expanding the tools available to chemical biologists. Parallel lines of enquiry (Phases A&B) will generate protein functionalization tools & synergistically feed into a Phase C, which will seek to translate the synthesis advances to application.

Access to novel disease-relevant protein and polypeptide constructs continues to be a challenge in academic laboratories, the pharmaceutical and SME biotech industries, potentially costing many lives and many £millions per year in healthcare investment and loss in productivity Protein modification is integral to chemical biology, molecular biology & biomedical science with applications that range from imaging to cell surface labelling & biocatalysis to drug development. The diversity of the proteome dictates that there is need distinct & complementary protein modification strategies. Synthetic design lies at the heart of new chemical methods for protein functionalization, however its challenges are very different from those in small molecule chemistry and requires a different approach. The EPSRC has prioritized Chemical Biology & Biological Chemistry for Fellowships. This proposal is also aligned to other themes of the portfolio such as Healthcare Technologies (in particular, new medicines) & Physical Science (Synthetic Organic Chemistry & Catalysis). In 2015, the UK had around 225 Biotech companies, turning over £2.9billion (an estimated 5% of the global market share) and employing almost 10000 people. Almost £1billion was spent on Biotech research in 2015 (4.5% of UK private R&D expenditure & the 4th largest global spender). By September 2018, UK Biotech companies had raised £1.5billion in funding - it is thriving & will continue to grow. The research outputs of this Fellowship could advance this sector through new technologies, new expertise & uniquely trained researchers, potentially new medicines and new commercial opportunities for spin out ventures.
An increasing focus on precision medicine and genetic understanding of disease will lead to a dramatic increase in the number of potent and highly selective molecular targets; identifying genetically informed targets could double success rates in clinical development (Nat. Gen. 2015, 47, 856). There is an urgent need for complementary and distinct chemistry that provides practical solutions to protein functionalization problems. Therefore, the development of new tools that rapidly assemble information rich protein constructs is a key challenge to the continued advance of chemical biology and drug discovery. The research could be crucial to the future prosperity of UK Pharma & biotech, as well as addressing unmet health needs. The proposal has a good fit with the EPSRCs portfolio in healthcare technologies and its emphasis on growing chemical biology. The PI is a world leader in synthetic chemistry, but through this Fellowship aims to established a synthesis-driven chemical biology program. The highly educated and trained synthetic chemists produced through this research program will be able to translate the outputs of this research through their future careers in research.