Hijacking prenyl and geranyl transferases - A route to carry out click modifications and to enhance cellular permeability of peptides

Recent advances in biological research have allowed a better understanding of the causation of many diseases and identified new targets for therapy. An ideal drug should bind to a specific cellular target and have no affinity to others. One of the recently identified challenging targets for drug discovery is the protein-protein interactions that have been proved to be involved in many difficult-to-treat diseases e.g. immune disorders and cancer. These interactions are taking place along the extended surface of large proteins and thus are very challenging for small molecule drugs. Biological drugs e.g. antibodies are large molecules and can disrupt protein-protein interactions but cannot be administered orally and are very expensive. Macrocyclic peptides are an emerging class of drug candidates that have the ability to disrupt protein-protein interactions e.g. the immune-suppressant, cyclosporin that made transplant surgery possible. They are smaller in size and are very much cheaper than biologics. In contrast to their linear "non-cyclic" counterparts, they are more stable against enzymes and are semi-rigid to fit better with their targets much like a key fits into a lock. A limitation that hampers the development of many of these compounds is their low ability to cross cellular membranes and to reach intracellular targets. Several modified cyclic peptides are commonly found in medicinal natural products. These compounds were evolved via natural selection which is presumably driven by their pharmacological potency against specific molecular targets as well as their ability to reach these targets that is, by crossing one or more biological membranes. In nature, several modifications are introduced to cyclic peptides to enhance membrane permeability. These modifications aim to reduce the hydrophilic (polar) surface of the molecule by shielding with hydrophobic side chains thus the compound can easily diffuse through the hydrophobic (mainly lipid) cellular membranes. Ideally these modifications should be applied to specific sites to avoid a large reduction in water solubility or the change of three dimensional shape of the molecule with subsequent decrease in its ability to bind to its target. Recent research revealed how a large group of these modified cyclic peptides is made inside their hosts. In this project, I will identify and recruit new modifying biosynthetic enzymes that will add hydrophobic chemical groups such as prenyl and geranyl groups at specific sites in cyclic peptides. Making these modifications using chemical methods is very challenging, not eco-friendly and in most cases entails total re-synthesis which is time consuming. I will determine the structure and biochemical features of these enzymes to identify the key residues that underlie their activity and specificity. I will use these insights to engineer and generate enzyme variants with different residue specificity and ability to introduce other chemical groups. I will use chemical synthesis and the engineered enzymes to generate modified derivatives of bespoke bioactive cyclic peptides and test the effect of different modifications on membrane permeability and the three dimensional shape of the molecule that underlies target affinity. These data will help to generate a computational model to predict membrane permeability of bioactive cyclic peptides that will be invaluable for development of peptides into drugs.

The proposed work has an impact on economy, society and environment.
Economic impact: The proposal will have an impact on industrial biotechnology and pharmaceutical industry sectors. Both industries are identified as crucial to the UK economy and are consistently at the top of industrial sectors in terms of trade surplus and number of employees, with around 73,000 people employed directly (Source: Association of British Pharmaceutical Industry). Currently, there is a global pharmaceutical interest in developing constrained / cyclic peptides as cheaper and equally potent alternatives to biologics. However, the poor ability of these compounds to cross cellular membranes has been identified as an impediment to their development as therapeutics. Introducing structural modifications to enhance permeability and balancing this with other physicochemical parameters that underlie pharmacodynamic and pharmacokinetic properties are very challenging and involve synthesis and testing of large number of variants which is time consuming. This project will give a market leadership to the UK as it provides novel enzymatic tools that can make challenging chemical modifications to peptides with high residue, regio- and stereo- specificities. Another important deliverable is a theoretical model to predict cellular permeability of cyclic peptides and modified derivatives. The project is multidisciplinary with elements of synthetic biology, peptide chemistry and theoretical chemistry. The EPSRC has considered synthetic biology of high strategic importance to the UK and has made a commitment to invest more in this area.
Societal Benefit: The project will benefit the society by providing trained workforce in biotechnology, synthetic biology and peptide therapeutics. School children and the general public will benefit from the planned outreach activities that will inspire children and increase the awareness of the public with synthetic biology. The project will provide novel enzymatic tools to modify peptides for enhanced cellular permeability and diversity. These compounds have underexploited potential in combating a range of diseases such as infection, cancer and autoimmune disorders. Introducing these modifications will allow exploring the potential of these compounds in tackling intracellular therapeutic targets and thus open new avenues for discovery and development of new drugs for critical diseases without currently available treatment options. The work will also generate a library of new biologically active molecules designed to disrupt key intracellular protein-protein interaction in Hippo signalling pathway involved in tumour development.
Environmental impact: The proposed research provides a green method to make challenging chemical transformations. The use of enzymes instead of toxic chemical reagents saves on energy required for production and is eco-friendly. In many occasions, enzymes can be immobilised and recycled and this saves on the amount of energy used in their production.