Feature: Tackling tritryps

The biology and business of drug development

Understanding the biology of the tritryps is suggesting ways they can be countered. But it may take novel commercial mechanisms to get drugs to patients.

The clamour for safe and effective drugs to combat tritryps is growing louder. In the 1960s, Chagas' disease, African sleeping sickness and leishmaniasis were largely under control. But in the last 25 years there has been an alarming rise in the number of cases. In several regions, these diseases re-emerged in epidemic proportions. There are no vaccines, and drugs are expensive, difficult to administer and toxic.

Despite the pressing need, these tropical diseases remain 'neglected' by modern medicine. There are no affordable, effective medicines available and the pharmaceutical industry has little incentive to develop new treatments. The process of bringing a drug to the market is expensive, and companies stand little chance of recouping their investment (see 'New ways to market' box below). As a result, drug development is almost at a standstill.

Academic scientists such as Professor David Barry at the Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, are determined to find ways round this deadlock. "We need to get to know the enemy," says Professor Barry, whose strategy hinges on uncovering the parasite's survival tactics. The idea is to find a protein or process that helps the parasite survive or infect its host, then to search for a tactic or chemical that acts on these targets.

Professor Barry has focused on the African trypanosome and the point at which it gets into the host. For the parasite, there is an element of uncertainty, as it does not know what host species the fly will feed on next: "The trypanosome must be adaptable enough to cope with a huge range of environments. It has to fight off the immune system, and it does so with antigenic variation."

The parasite has a surface coat made out of complex molecules called variant surface glycoproteins (VSGs). "If the host makes antibodies against these surface coats, that is the end of the parasite," says Professor Barry.

To avoid being wiped out by antibodies, the parasite switches to a new surface coat. "The trypanosome has some very clever ways of generating that diversity," says Professor Barry. It is equipped with more than 1000 genes coding for VSGs, only one of which is active at any one time. Every so often, the parasite switches the active VSG gene. It uses a remarkable mechanism, whereby the old gene is ejected from the active area of chromosome, and a new one is inserted, like changing CDs in a CD player.

This switch happens about once every 100 divisions. The parasite with the new coat survives the antibody onslaught while the previous population is wiped out. The survivors then grow and a new cycle begins.

"Antigenic variation has a lot to do with the way the way the parasite grows in the host," says Professor Barry. "We don't see the parasite overwhelming the host, growing out of control because there is a moderation of its growth imposed by this constant peaking and knocking down."

VSG genes have been intensively studied, but the genome sequence has changed thinking quite substantially. "The genome sequence project is telling us that the scope for antigenic variation is potentially far greater than we suspected."

Far from being disillusioned, Professor Barry is enthused. "It is critical that we understand the role of this system in the field, to help us comprehend transmission, not just of trypanosomes, but also other parasites. Furthermore, by allowing the possibility of comparison with other, better studied organisms, the genome sequence is helping in the search for molecules involved in control of surface coat expression."

Closing the pharma gap

At the University of Dundee, Professor Mike Ferguson is focused on finding critical molecules within the parasite's architecture. "If we know that a molecule is essential for the infectivity of the parasite, then we can find a drug that prevents these surface molecules from being assembled – that would be a useful drug against the disease."

Professor Ferguson has already pinpointed one molecule worth targeting: an epimerase enzyme of T. brucei. The parasite's surface coat proteins are studded with a complex arrangement of sugar molecules, including galactose. But the parasite cannot take up galactose. Instead, it converts glucose into galactose during the construction of the surface coat – a task carried out by an epimerase. With the enzyme out of action, the parasite fails to assemble its coat and dies.

The first task was to identify the gene that encodes the enzyme. "This is where we use the genome data as a library. We have to mine out the relevant information," says Professor Ferguson (see 'Quickening the pace' box). The second task was to use gene-knockout methods to prove that the gene was essential. Since then, Professor Ferguson's colleague, Professor Bill Hunter, has resolved the structure of the enzyme by X-ray crystallography. Now the search is on for inhibitors as drug leads.

Another of Professor Ferguson's targets is the glycosylphosphatidylinositol (GPI) biosynthetic pathways in T. brucei. The GPI molecule keeps cell surface molecules anchored; if GPI synthesis is disrupted, it produces a vulnerable, naked parasite. "It has great potential as a broad specificity anti-parasitic agent that will hit a variety of diseases: African sleeping sickness, Chagas' disease, Leishmania and malaria." The Ferguson lab has developed molecules that can get into cells and spike the GPI pathway, but are not toxic to people – proof that this approach could work in practice.

But while basic science research can throw up promising drug target candidates, there is still a leap to actual drugs entering clinical trials. Filling this gap has been traditionally in the hands of the pharmaceutical industry. Sadly, tropical diseases are not attractive to industry (see 'New ways to market' box).

"In Dundee we intend to go one step further – we intend to progress drug targets that show the most promise through to actual drugs," says Professor Fairlamb. To propel promising leads into a drug pipeline, he and others are setting up a Centre for Interdisciplinary Research at the university. The purpose is to use high-throughput screening, a technology normally run only by large pharmaceutical companies, to find promising drug leads. A robotic screening system will allow the Scottish team to can test 100 000 compounds against chosen targets.

"This initiative will be unique in the whole of Europe," asserts Professor Ferguson, who expects the centre to be up and running by the summer.

Leishmania leads

"At the time that we started work on Leishmania, we hadn't even thought of sequencing the genome," says Professor Debbie Smith, at the University of York. "Fifteen years later, genomes are coming out, and we can accelerate our post-genomic research."

Most of Professor Smith's efforts have been directed at identifying proteins expressed during the parasite's infective stages, which is when they cause disease in people. The most interesting protein pulled up by Smith's lab is HASPB, which is proving to be a good vaccine candidate (see Fighting back).

But studying HASPB had an unexpected off-shoot: a novel target for chemotherapy. The researchers were drawn towards the enzyme that attaches acyl groups onto the protein, N-myristoyl transferase (or NMT). While not unique to the parasite, it is sufficiently distinct from the human enzyme that any drug designed to stop its function would not affect mammalian cells.

And the story gets better. Some years ago, NMT was touted as a promising drug target for pathogenic fungi and, at the time, pharmaceutical companies developed a clutch of NMT inhibitors. But, despite being potent inhibitors, these compounds failed as anti-fungal drugs, because they were specific for each type of fungus. So the compounds were dropped.

But big pharma discards may be just what Professor Smith is after. "Half the development work has been done. There are already banks of inhibitors that we can use." Pfizer is giving away these inhibitors to the York team, which has shown that they do inhibit the enzyme in T. brucei.

Using genomics tools, Professor Smith is searching for targets downstream of NMT. "We can pull out those proteins from the genome databases and we can have a whole range of them." Once the genes have been identified, the group can knock out each gene downstream of NMT using RNA interference and find out whether they are essential to the parasite. "That work is allowing us to understand how parasites are killed, [to know] which downstream targets are inhibited."

Professor Smith hopes that academic research will soon translate into practical solutions. "These diseases are on the increase and there are no effective therapies. It's very important to get a handle on them."

New ways to market

For Professor Alan Fairlamb, discovering a chink in the parasites’ armour was a watershed. In 1985 he discovered trypanothione, a molecule that is unique to tritryps. He immediately recognised that this molecule would make an ideal drug target, and began a collaboration with GlaxoSmithKline.

“We assumed that, having made these fundamental discoveries, pharmaceutical companies around the world would take up this challenge and turn this basic knowledge into treatment for these neglected diseases,” recalls Professor Fairlamb, a Wellcome Principal Research Fellow at the University of Dundee.

The attraction of trypanothione as a drug target is that it is found in all the tritryps, the cause of three major diseases: leishmaniasis, African trypanosomiasis and Chagas’ disease. All three parasites rely on trypanothione to protect them from the ravages of oxidative and chemical stress. If scientists can design a drug to inhibit it, the parasites will perish.

Twenty years on, despite strong genetic and chemical evidence of the value of trypanothione reductase as a drug target, the drug has proved elusive. “Pharmaceutical companies frequently dismiss these neglected diseases as unprofitable for drug development,” Professor Fairlamb explains. “They are interested in blockbuster drugs and inevitably that means that diseases of the developing world become uneconomic.”

Yet his belief in trypanothione as a druggabl etarget is unwavering. “The fact that GSK validated my claims – that this was a suitable target for development – means that two agencies have now funded screens elsewhere.” In addition, his team has characterised several other promising potential drug targets, focusing on other aspects of trypanothione metabolism.

The need for new therapies is as urgent as ever, but Professor Fairlamb is now taking a different tack. “The prospects of turning basic research discoveries into practical products is moving forward, this time without the input of pharmaceutical industry,” he says. The University of Dundee is establishing a high-throughput screening centre with medicinal chemistry facilities in a new £18.5 million Centre for Interdisciplinary Research. “I’m optimistic about the future, but realistically, I’m aware it will take another five to ten years to get products into clinical trials.”

Quickening the pace

For researchers, the genome sequence is all about acceleration. In Professor Mike Ferguson’s lab, it has allowed them to identify proteins starting from tiny snippets of information, or ‘tags’. “You can only do that if you have all the genes available for screening,” adds Mike Ferguson, who heads the team. “That was impossible before the genome was sequenced,” he says.

The epimerase gene, for example, was identified in a search through the trypanosome database, starting with the sequence of the human version of the gene. As a result, the gene was cloned in record speed. “If we’d done that without the genome data, it would have probably taken us five years of biochemical slog. But because the genome data were sitting there it took three months.”

The world needs new drugs

Drugs to treat sleeping sickness are old and toxic. The drug melarsopol, an arsenic derivative, has been in use since 1949. Without melarsopol, sleeping sickness is fatal, but the treatment itself kills one in 20. Eflornithine is a more recent drug, which is less toxic but needs to be injected four times a day.

Leishmaniasis treatment depends on toxic antimonial drugs that need to be infused parenterally, while in hospital.

The first pill for visceral Leishmania, miltefosine, has been available since 2002. The drug is 95 per cent effective at curing the disease, but causes birth defects, so women of childbearing age must use contraception.

Few drugs are available to treat Chagas’ disease. Spraying of houses that harbour the insect vector is the usual control strategy.

Image: Professor Dave Barry of the Wellcome Trust Centre for Molecular Parasitology at Glasgow, courtesy of the Wellcome Trust Medical Photographic Library.

Feature: Tackling tritryps

The biology and business of drug development

Closing the pharma gap

Leishmania leads

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