Australasian Science: Australia's authority on science since 1938

Pharmed Meds

Credit: Maksym Yemelyanov/adobe

Credit: Maksym Yemelyanov/adobe

By Karen Harris & Marilyn Anderson

Some clever chemistry is turning plants into pharmaceutical factories that could enable remote communities in developing countries to grow and store stable medicines cheaply.

The distribution of medicines in developing countries is a logistical and economic challenge. Medicines must be cheaply produced so that they are affordable for people with little income, and then they must be maintained in an active form throughout transport and storage.

An enticing and economical alternative to lab-based pharmaceuticals is to harness plants to produce orally active edible meds. Taking your meds could be as simple as eating your greens or sprinkling some sunflower seeds on your breakfast cereal. However, this dream can only become a reality if plants can be engineered to consistently manufacture medicines in an orally available form.

Proteins and peptides meet the first of these criteria: they can be readily engineered in plants because their genetic code can be manipulated to produce a given amino acid sequence. Protein-based medicines also offer a larger surface area to interact with their targets, leading to greater specificity and fewer off-target effects. Protein-based pharmaceuticals currently on the market include synthetic insulin to treat diabetes and the anti-HIV medication enfuvirtide.

However, proteins generally don’t meet the second criteria. They are often ineffective when administered orally because they aren’t stable in biological fluids and are rapidly broken down into inactive fragments before they reach their target.

Tantalising early evidence that orally available peptides could be delivered in plant material comes from traditional medicines. In the 1960s, Norwegian doctor Lorents Gran observed that women in the Democratic Republic of the Congo used the leaves of a native plant, kalata-kalata (Oldenlandia affinis), to produce a medicinal tea that was given to women in childbirth to accelerate labour. The active ingredient was incredibly stable and remained orally available even after boiling. This was soon identified as a 29 amino acid peptide, but it was some years before the structure of this peptide was solved, revealing a rigid set of cross-links as well as a circular backbone that provided a molecular basis for its incredible stability.

The circular nature of this peptide was particularly unusual since peptides are more typically linear, with distinct termini. The circular peptide backbone contributes to its stability by introducing structural constraints while making the ends of the peptide inaccessible to proteases that degrade peptides into smaller parts. Circular peptides of this family, named cyclotides, have subsequently been isolated from numerous plants and ascribed a range of bioactivities against insects, molluscs, nematodes and HIV. Cyclotides can also be engineered to exhibit other functions.

The rigid core of cyclotides is surrounded by loops that can be modified. Grafting of new bioactive peptides within this scaffold can improve the oral availability of otherwise unstable peptides. The most recent example of this was the engineering of a cyclotide scaffold to impart specific activity against receptors involved in inflammatory pain. This new peptide was orally available in mouse models.

Circularisation can also been applied to peptides that are naturally linear. This has successfully increased the potency, oral availability and selectivity of a linear peptide derived from the venom of cone snails. The circularised peptide was 120 times more potent than gabapentin, a drug in clinical use for the treatment of neuropathic pain.

These examples highlight the excellent potential of both cyclotide scaffolds and the more generally applicable circularisation of peptides to produce potent, stable drugs.

Cyclic peptides are efficiently produced in high amounts in their native hosts and are orally available from plants. However, the efficiency of cyclotide production is not replicated when cyclotide genes are expressed in other types of plants. Yields in these transgenic systems are poor and the linear version of the peptide dominates. It’s likely that plants that do not naturally produce cyclic peptides do not have the right machinery for this modification.

To develop strategies to overcome poor yields in engineered plants we returned to kalata-kalata, the prototypic cyclotide-producing plant, to search for the native processing machinery involved in cyclotide maturation. This led to the identification of an enzyme called an asparaginyl endopeptidase, which processes cyclotide precursors to the native circular peptide. We have produced this enzyme in the laboratory and shown that it converts precursor peptides to their cyclised form with almost 100% efficiency. This is applicable not only to native cyclotides but also to a range of structurally and functionally unrelated linear peptides. This means that circularisation of essentially any target peptide is likely to be rapidly achievable using this strategy.

The next stage of development of this exciting finding is to use this technology to create transgenic plants that contain both the target peptide and the machinery for circularisation.

While the co-introduction of these two components will overcome the efficiency limitations previously observed in transgenic systems, a number of challenges will still need to be addressed before this system can be adopted for the production of pharmaceuticals. Regulation of dose is one such obstacle, and careful evaluation of transgenic plants under well-controlled conditions will be required to determine the reproducibility of yields.

For this to be of most use to the developing world it will be crucial to achieve consistency in the field, outside of the controlled greenhouse environment. A further advantage of protein-based drugs is that they are readily quantifiable. Ideally, a simple colour-based assay would enable locals with little technical expertise to measure doses quickly and accurately for each therapy.

A further challenge to implementing edible medicines will be education. Fear surrounding genetically modified foods remains a reality despite the weight of evidence indicating that GM foods are not harmful. Furthermore, a therapeutic agent ingested directly from plants will be subject to the same rigorous quality control and safety profile required for the approval of any drug.

Harnessing the native machinery for circular peptide production in plants will also allow the development of plant-based systems to produce, extract and purify circular peptides under the strictly controlled conditions required for the production of pharmaceuticals. This alternative to established methods is already in operation for other drugs, providing a fast, scalable and environmentally friendly means of pharmaceutical production.

For example, drugs that target the Ebola virus and vaccines against influenza have been rapidly produced on a large scale in tobacco plants and extracted in a pure form. This approach is attractive for cyclic peptides since achieving the circularisation step in vitro has in the past been challenging and expensive, limiting large-scale application.

Finally, the production of the enzyme responsible for efficient circularisation represents a major breakthrough in itself. In addition to the plant production system, peptides could be produced in bacteria or yeast, and then processed using the native machinery to their circular forms in the laboratory. As this eliminates the need for researchers to have technical knowledge about plant transfection and peptide synthesis, it would likely fast-track the application of this modification to other lead peptides in drug design.

Growing plants that will naturally produce medicines that are orally available is the Holy Grail of drug delivery. The favourable pharmacokinetic profile of circular peptides offer a unique opportunity for achieving this. Targeting these orally available medicines into edible components of plants in varieties that are easily grown in a broad range of climates and conditions will offer a viable, economical alternative to the traditional production of pharmaceuticals and redefine the distribution of medicine to developing countries.

Dr Karen Harris is a research fellow supervised by Prof Marilyn Anderson of La Trobe University’s Department of Biochemistry and Genetics.