Proteases are a class
of proteins that function by catalyzing the cleavage or breakdown
of other proteins1,4.
This cleavage is often necessary to activate, inactive, degrade
or modify proteins for their proper function in the body, and
is an important regulatory system in living cells. Hydrolytic
cleavage of peptide bonds usually occurs in a predictable and
reproducible way at specific sequences of amino acids that are
recognized by a given protease. Many proteases are very specific
and produce a limited proteolysis; for example, trypsin is a protease
that cleaves after lysine and arginine residues. Other proteases
degrade proteins completely into amino acids by cutting at many
different sites1,4.
Proteases are essential to natural physiologic processes such
as inflammation, infection, fertilization, allergic reactions,
cell growth and death, blood clotting, tumor growth and bone remodelling1.
Many diseases are a result of protease misfunction: proteins that
are no longer needed may fail to be disposed of properly, or important
proteins needed at a specific time never get activated or are
degraded before they can carry out their function. All these scenarios
can result in cellular malfunction that can lead to a variety
of diseases4,6.
Matrix metalloproteinases
(MMPs) are a class of proteases that have a role in cancer. In
normal physiology, MMPs are produced by connective tissue and
are thought to contribute to tissue remodeling in development,
in the menstrual cycle, and as part of repair processes following
tissue damage. However in cancer, aberrant MMP activity can degrade
the extracellular matrix that surrounds cells allowing tumours
to invade surrounding tissue and spread the tumour to other sites
in the body. They are also involved in promoting metastasis and
angiogenesis (the formation of blood vessels around the tumour
that feed it, allowing it to grow)2,5,6.
In HIV/AIDS, the HIV protease encoded by the HIV virus plays a
critical role in the virus life cycle. The proteins that make
up the HIV virus particles are produced as long "polyproteins."
These precursors must be cleaved to yield the active proteins
of the mature virus. The HIV-1 protease is an aspartic protease
(meaning it cleaves after aspartate residues) that functions to
cleave the nascent polyproteins during viral replication to allow
assembly of mature virus particles. The protease is only expressed
in infected cells, making it a good target for a drug that inactivates
the protease but leaves other cellular proteins intact. Many of
the available drugs for AIDS are inhibitors of this protease.
However, inhibiting the protease alone has not resulted in eradication
of the virus. HIV can quickly evolve resistance to the drugs by
changing the amino acid sequence of the protease through DNA mutations,
which occur at high frequency in this virus3.
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Figure
1. Protease activated toxins. |
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Twinstrand is exploiting
the unique properties of proteases in a different approach to
treating disease. It has taken advantage of the importance of
these proteases by engineering a series of potent plant toxins
that are activated by cleavage by specific proteases. These toxins
when first produced are in a precursor, inactive state. In their
natural form, the toxins get activated by plant proteases that
cleave at a specific location, revealing the active domains of
the toxins and ultimately resulting in toxin-mediated killing
of the cell (7). The basis of Twinstrand's
research is to substitute these natural, plant-specific protease
sequences for sequences that are instead recognized by proteases
associated with progression of certain diseases. By inserting
the cleavage site for an MMP protease associated with cancerous
cells, for example, cleavage of the sequence will result in an
active form of the toxin being generated by the MMP within the
cancerous cells, but not in surrounding healthy cells that do
not express or express only very low levels of the MMP protease7.
The toxins Twinstrand
uses are class II ribosome inhibiting proteins (RIPs) (for example,
ricin) - toxins that, when activated, disrupt protein synthesis
in the cell, quickly resulting in cell death7.
An advantage of this
technique is that the drug's mechanism of action is very different
from traditional chemotherapy drugs. This means that the two treatments
are unlikely to interfere with each other and could be used in
combination therapies. Furthermore, the toxin treatment could
be important in treating tumours that have become resistance to
conventional chemotherapy drugs. The treatment is also not genotoxic
(does not cause permanent damage to DNA of surrounding cells),
and animals that were exposed to sublethal doses of either the
natural toxin or the modified protease target sequence toxin recovered
from the treatment with no long term effects7.
Cancer: Twinstrand
is using proteases associated with tumours to activate the RIP
toxins. These include matrix metalloproteinases MMP-2 and MMP-9.
Twinstrand has designed plant toxins that can be specifically
activated by these two proteases. Research has shown anti-tumour
activity of the MMP-cleavable toxins in rodents. Other cancer-associated
proteases that have been developed for recombinant toxin targeting
are MTI-MMP, uPA (urokinase plasminogen activator), tPA (tissue
plasminogen activator), cathepsin B (intracellular cysteine proteases
involved in diverse activities such as blood clotting, cancer
growth and metastasis and bone remodeling) and prostate specific
antigen1,7.
Other diseases that
involve specific proteases could also potentially be targeted
with this technique. They include HIV/AIDS, Hepatitis A and C,
cytomegalovirus, herpes, parasitic infections (malaria, schistosomiasis),
fungal infections, and inflammatory diseases (e.g. Arthritis)1,7.
Twinstrand is also
using a proteomics-based strategy to discover new proteases and
protease inhibitors specific to healthy and diseased tissues.
This may lead to the identification of new drug targets in other
diseases7.
