Methods for bioengineering NRPS/PKS assembly lines delivering peptide natural products with electrophilic warheads.

Natural products (NP) are molecules isolated from microorganisms and plants that inspired the development of many leading antibiotics, anticancer, immunosuppressive agents and other essential medicines that are widely used in the clinic today. Often the NP that are isolated from the native organism do not possess the prerequisite properties at the outset. Further synthetic modification is typically required to provide the final drug compound. However, NP are typically highly complex molecules requiring laborious multistep chemical synthesis, which is very expensive, polluting and increasingly unsustainable. The difficulty associated with the synthesis of optimised NP variants presents a major barrier to pharma companies undertaking drug development. This is particularly problematic in the manufacture of drugs required to treat diseases of developing world such as malaria, Leishmania and Chagas disease. These highly infectious diseases, caused by single celled (protozoan) parasites transmitted by insects, effect billions of the poorest people in the world and lead to over 500,000 deaths pa. Currently there are very few effective treatments available for these diseases. A NP artemisinin is used to treat malaria, but new strains of the malaria parasite (P. falciparum) have emerged which are resistant to artemisinin. Promising new NP leads have been identified for malaria and other related diseases, but the costs of synthesising derivatives have prevented new treatments being made available. An alternative for producing optimised NP derivatives, is to manipulate the biosynthetic assembly lines (enzymes) in the microorganisms that construct the parent NP. By reprogramming (engineering) the assembly line to accept different precursors, NP variants with improved properties can be delivered in a more efficient, cost-effective single-step fermentation process.

In this project we aim to engineer biosynthetic pathways to produce NP derivatives with antiprotozoal activity that could be used to combat malaria or related diseases. The target NP are peptides, composed of amino acids with a reactive terminal functional group (warhead), produced by Streptomyces and other bacteria. These NP will be designed to bind to the proteasome of protozoa such as P. falciparum. Proteasomes are large multi-protein complexes responsible for degrading other proteins in the cell that are either damaged or no longer needed. The warhead of the peptide NP can cross-link with the proteasome inhibiting its function leading to cell death. We will use novel gene editing and other approaches to engineer the genes encoding the enzymes that assemble the warhead containing peptide NP. This will allow us to change the sequence of the peptides and also include different warheads, to improve their activity, selectivity and other properties for drug development. The biosynthetic assembly line includes nonribosomal peptide synthetase (NRPS) enzymes that condense amino acid precursors. By replacing domains, or subdomains, within the NRPS it is possible to change the sequence of the amino acids in the peptide products. Guided by earlier synthetic studies, we will create warhead containing peptides that are highly selective for the P. falciparum proteasome. Compounds that inhibit proteasomes in human as well as the parasite cell, would be toxic and unsuitable. We will also engineer assembly lines that deliver warhead containing peptides designed to inhibit the proteasomes in human cancer cells. This includes oprozomib a synthetic analogue, which is in clinical trials for treatment of multiple myeloma (bone marrow cancer). By developing an engineered pathway to this type compound, it may be possible to produce anticancer drugs, like oprozomib, in a single-step fermentation making them more widely available at lower costs. The methods we develop are generic and can be used to produce a range of warhead containing peptides for a number of other therapeutic applications.

Natural products (NP) have inspired the development of a high proportion of important pharmaceuticals in use today. Many of the most promising NP are assembled by nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and hybrid NRPS-PKS enzymes. NP derived from these mega-synthase enzymes are often complex structures requiring multi-step synthesis to generate optimised derivatives, which is costly, polluting and offers limited opportunity for commercial development of new drugs. Alternative bioengineering approaches provide more sustainable and cheaper routes to optimised NP variants via a single-step fermentation. The recent explosion in publicly available genome sequencing data coupled with developments in structural biology and synthetic biology have afforded new insights and tools that can be employed to rapidly engineer pathways, including mega-synthases, to deliver optimised targets.

Recently we have discovered and characterised a number of NRPS-PKS enzymes, including the the mega-synthase that delivers epoxyketone (EPK) proteasome inhibitors. EPK NP inspired the development the anti-cancer drug carfilzomib. Here we aim to combine our knowledge of EPK biosynthesis with new methods for reprogramming mega-synthase enzymes, in order to deliver EPK variants that are selective proteasomes of protozoal pathogens including the malaria parasite. We will engineer the NRPS-PKS from EPK pathways to deliver peptides including epoxyketone, aldehyde and vinylsulphone warheads, which are highly selective for Plasmodia proteasomes. In addition, we will reprogramme EPK assembly lines to deliver products closely related to oprozomib, a second generation orally active EPK, which is currently in clinical trials for treatment of multiple myeloma. The methods we develop can be used to produce a raft of warhead containing peptides which could be further developed to target proteases, as well as proteosomes, which are implicated a wide range of diseases.