Feature: Turning toxins against MS

This feature appeared in the September/October 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

The waving tentacles of the Caribbean sun anemone, Stichodactyla helianthus are tipped with stinging nematocysts that pack a paralytic punch: a cocktail of neurotoxic and haemolytic peptides that rapidly immobilise any small reef fish or crustacean that blunders into them. The anemone's array of peptide toxins includes ShK (S. helianthus, K-channel), a 35-residue molecule that blockades voltage-gated potassium channels on nerve and muscle cells, leaving prey twitching helplessly in the anemone's embrace.

Swedish researchers isolated ShK from the venom of the Caribbean anemone in the early 1990s and found it to be a generic potassium-channel blocker. Yet, intriguingly, the fish- and crustacean-paralysing peptide has emerged as one of the most promising experimental therapies for the debilitating human autoimmune disorder multiple sclerosis (MS).

Professor Ray Norton's research team at the Walter and Eliza Hall Medical Research Institute has spent the past decade investigating ShK's structure and function, and developing analogues as potential therapies for auto-immune disorders. Norton will describe his team's research to the Australian Peptide Association's 8th Australian Peptide Conference on Stradbroke Island in October.

ShK's therapeutic promise rests on its ability to blockade a particular potassium channel sub-type found on the surface of effector memory T cells (TEM cells). TEM cells help direct the destructive immune-system assault on the 'self' antigens involved in MS and several other auto-immune disorders involving T cells, including rheumatoid arthritis and Type 1 diabetes. During quiescent phases between attacks they maintain a molecular memory of the target auto-antigens.

Norton says TEM cells activate when receptors on their surface contact the target auto-antigen, triggering a sharp rise in intracellular calcium ions. The calcium influx induces a voltage rise at the cell membrane, which opens Kv1 channels and allows potassium ions to flow out of the cell, restoring the ionic balance – thus the term 'voltage gated'. The transient calcium pulse activates the T cell, causing it to begin proliferating. Norton says that selectively blockading the potassium channels that trigger activation maintains the TEM cells in a quiescent state, halting the attack on tissues displaying the auto-antigen.

In multiple sclerosis, autoreactive cytotoxic T cells attack the insulating sheath of myelin that wraps around the axons of neurons in the brain and spinal cord, preventing current leakage as action potentials propagate through neural networks. The target in MS is myelin; in Type 1 diabetes, it's pancreatic islet cells or insulin; and in rheumatoid arthritis, joint-cushioning cartilage.

In MS, waves of T cells attack the myelin sheath, causing lesions that expose the nerve cell axon and cause current leakages, short-circuiting the transmission of nerve signals. In severe cases, paralysis results. Blocking the potassium channels that activate TEM cells maintains them in a quiescent state, halting the cycle of attacks on the central nervous system at its source.

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Toxin to the rescue

Norton says the sun anemone's ShK peptide weakly binds most Kv1-type potassium channels, but exhibits moderate selectivity for Kv1.3 channels. In humans, Kv1.3 channels are mainly expressed not by nerve cells, but by the TEM cells that initiate the auto-immune response. Blocking Kv1.3 channels on TEM cells thus offers a way to arrest all TEM-cell-mediated auto-immune disorders, with minimal side effects.

"Kv1.3 channels are not restricted to TEM cells, but they're not found on many other cells," says Norton. "In principle, Kv1.3 blockers have the potential to block TEM-associated autoimmune disorders without causing the generalised immunosuppression associated with drugs like cyclosporin."

The prospect that the sun anemone's ShK neurotoxic peptide could be the prototype for a generic therapy for an entire class of auto-immune disorders has motivated a decade-long collaboration between Norton's Walter and Eliza Hall Medical Research Institute group and Professor George Chandy's research team at the University of California, Irvine, and Dr Mike Pennington at Bachem in Philadelphia.

Norton says Chandy's team has been conducting electrophysiological studies to determine how well – and how selectively – candidate peptides suppress activation of TEM cells. The UC Irvine researches have also assessed candidate therapeutic peptides in mice with experimentally induced auto-immune encephalomyelitis (EAE), an animal model of human multiple sclerosis.

Since determining the three-dimensional structure of ShK a decade ago, Norton and his collaborators, including Dr Brian Smith from the Hall Institute, have developed models of its molecular 'fit' with various Kv1-type potassium channels. Their efforts have identified the peptide elements that underpin its generic ability to blockade Kv1 channels.

All Kv1-type potassium channels are tetramers, with the four subunits arranged to form a central channel traversing the cell membrane. The subunits relax and constrict in response to changing electrical potentials at the cell membrane – hence 'voltage-gated channel'. They form a bi-directional valve that regulates potassium concentrations in the host cell. Kv1.3 homotetramers, made up of four identical Kv1.3 subunits, yield the best 'fit' with ShK, and explain the peptide's moderate natural selectivity for Kv1.3 channels.

Unnaturally selected

But natural selection's best fit is not always optimal for safe, effective therapeutic use in humans. Norton, Chandy and Pennington have spent several years experimentally substituting critical residues in the ShK peptide to increase its selectivity for Kv1.3 channels. By attaching an adduct to the N-terminus of a peptide with substituted residues, they succeeded in designing two candidate analogues with exceptional selectivity for Kv1.3 channels, and correspondingly reduced avidity for other Kv1 channels. ---PB---

"It's a really tight binder – we're talking about picomolar affinities," says Norton. "Attaching the N-terminal adduct greatly reduced its affinity for other Kv1 channels, at the cost of only a slight reduction in its avidity for Kv1.3."

Norton also found an ingenious way to make the Kv1.3 analogue resistant to proteolysis, and potentially invisible to the immune system, which are potential obstacles for any peptide drug. All amino acids, with the exception of glycine, come in two mirror-image forms, called L- and D-enantiomers. The vast majority of life forms on Earth assemble their proteins and peptides from L-amino acids. However, reasoning that the target channel was a symmetric homotetramer, Norton substituted D-amino acids for the naturally occurring L-amino acids at every available residue in the chain. Because mammalian proteolytic enzymes are adapted to targeting L-amino acid peptides, the 'flipped' peptide should be resistant to proteolysis when used therapeutically in humans.

"Proteolysis should not be an obstacle to maximising the half-life of this analogue in human plasma," Norton says. "The half-life of ShK in plasma is relatively short – around 30 to 40 minutes – which would normally require patients to be continuously infused by the drug with something similar to the insulin pump that diabetics use."

But serendipity again intervened. In rats, the Kv1.3 analogue maintains its blockade on Kv1.3 potassium channels much longer than would be expected from its brief half life. "The peptide is eliminated renally, but it seems that for any cell with Kv1.3 receptors, some as-yet unidentified reservoir continues to maintain the blockade well after it should have been eliminated. In our rodent model of multiple sclerosis, we need to inject only once every 24 hours to obtain complete resolution of symptoms. At picomolar concentrations, ShK analogues modified at the N-terminus completely block Kv1.3 channels on TEM cells. The cells cease to respond to the antigen and stop proliferating. The peptide doesn't make the TEM cells forget the antigen, it just prevents them reacting to its stimulus."

Seattle-based company Kineta has signed an agreement with the research partners to take one of the candidate molecules into pre-clinical trials. It's early days, but the peptide's novel and highly selective ability to modulate the immune system makes it worth watching.