their native hosts and are orally available from plants. However,
the eiciency 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
modiication.
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 identiication
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% eiciency. 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 inding 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 eiciency limitations previously observed in trans-
genic systems, a number of challenges will still need to be
addressed before this system can be adopted for the produc-
tion 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 ield, outside of the
controlled greenhouse environment. A further advantage of
protein-based drugs is that they are readily quantiiable. Ideally,
a simple colour-based assay would enable locals with little tech-
nical expertise to measure doses quickly and accurately for each
therapy.
A further challenge to implementing edible medicines will
be education. Fear surrounding genetically modiied 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 proile required for the approval of
any drug.
Harnessing the native machinery for circular peptide produc-
tion 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 envi-
ronmentally friendly means of pharmaceutical production.
For example, drugs that target the Ebola virus and vaccines
against inluenza 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 circularisa-
tion step in vitro has in the past been challenging and expen-
sive, limiting large-scale application.
Finally, the production of the enzyme responsible for eicient
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 knowl-
edge about plant transfection and peptide synthesis, it would
likely fast-track the application of this modiication 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 proile 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 redeine 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.JUNE 2016|| 25emelyanov/adobe