Dengue and TB

The dengue research program at NITD has incorporated many of the most advanced drug discovery tools currently available: from structural biology based on three-dimensional crystal structures of target proteins, to generation of novel disease models and use of fragment-based screening in parallel with high-throughput screening of more than a million compounds from Novartis chemical archives. Moreover, Novartis has forged a web of academic collaborations to fill gaps in knowledge of biology underlying the disease – particularly the reasons that a relatively small proportion of cases escalate from a simple fever to the potentially life-threatening form, dengue hemorrhagic fever.

Prof. Martin Hibberd from the Genome Institute of Singapore spoke on the Genomics of Dengue.

Since viral load often is declining when dengue fever progresses to the more severe hemorrhagic form, even potent and selective drugs may not have a significant impact on the course of disease. Novartis and academic partners are racing to determine the role of viral load in progression to the hemorrhagic form of dengue – and whether rapid diagnosis and treatment at an early stage of infection will be a precondition for successful treatment.

So far NITD scientists have focused their target identification efforts on a handful of key viral enzymes that play critical roles in the disease. One of the enzymes, for example, enables entry of the dengue virus into the host cell and fusion of viral and host cell membranes. Other enzymes – including the NS3 protease, NS5 polymerase and NS3 helicase – are essential for viral replication. High-throughput screening programs for NS3 and NS5 targets have been completed and lead identification activities are ongoing. The best compound emerging from screening to date shows activity against the NS5 polymerase and has advanced to the lead optimization phase of drug discovery.

Dormant TB and the Global Challenge of Health

Progress of research against drug-resistant TB has been slower, reflecting the phenomenal capacity of Mycobacterium tuberculosis, the cause of TB, to adopt a dormant state and hide in the body, impervious to drugs, for months, years or even decades. If a host’s immune system is weakened, for example as a result of HIV infection, those latent bugs suddenly come out of hiding and reactivate TB.

Dormant TB bacilli dramatically reduce energy consumption and store supplies of energy in membranes and one hypothesis by NITD scientists is that disrupting those finely balanced energy production processes could render dormant bugs vulnerable to therapy. Systematic “knockout” experiments have identified dozens of genes in M. tuberculosis that are essential for survival; and screening efforts are ongoing to determine if inhibition of the function of those genes merely inhibits growth, or can actually kill the bacterium.

No new drug against TB has been launched for more than 30 years and the resumption of active research posed challenges for NITD scientists. “It turned out that important infrastructure – and many of the key research tools for TB – are still missing,” said Thomas Keller, Deputy Director and Head of Chemistry at NITD. “And our current chemical libraries may not be well-suited for TB drug discovery.”

Nevertheless, NITD has been tapped as the industry partner in the Grand Challenges in Global Health initiative launched by the Bill & Melinda Gates Foundation and the US National Institutes of Health. NITD will participate in a project, led by Professor Douglas Young of Imperial College London, aiming to discover drugs for treatment of dormant TB infection.

Fighting Malaria

In December 2004, Dr. Matter met in his Singapore office with representatives from the London-based Wellcome Trust and MMV, a not-for-profit organization based in Geneva that is dedicated to developing safe, effective and affordable antimalarials. The visitors urged NITD to consider moving into malaria.

There was a compelling case for NITD to join the fray, building on contributions Novartis made over the past decade in transforming malaria treatment with the pioneering medicine Coartem. “Coartem has become the current standard of malaria treatment – but we knew resistance to it could emerge,” Dr. Herrling said. While Coartem is the first artemisinin-based combination therapy, combining a synthetic chemical compound with a derivative of artemisinin, one of the most potent killers of the malaria parasite discovered to date, other artemisinin-derived drugs are available and some are sold as monotherapies. “Monotherapies can increase the probability of building resistance,” Dr. Herrling said. “We were concerned doctors and patients might eventually be left with no tools to fight malaria.”

NITD has assembled a broad portfolio of early-stage projects by forging research collaborations with academic researchers around the world.

Dr. Matter also had a longstanding interest in malaria – but he remained wary of diluting the productivity of NITD by focusing on additional diseses. So he pressed the visitors from Europe to describe their “absolute dreams” for a next-generation therapy. “Once I managed to pry that out, everything fell into place,” he recalled with a smile.

A single-dose cure for P. falciparum malaria headed the wish list of MMV President and CEO Chris Hentschel. The dream of Ted Bianco of the Wellcome Trust was a curative modality for P. vivax, a form of malaria that has expanded rapidly in recent years. “For our new partners, the focus, infrastructure and solid expertise of NITD were very attractive,” Dr. Matter said. “Still, some people told us we were totally crazy and that we’d never achieve those ambitious goals.”

Global Malaria risk levels

Click to enlarge

Confounding the skeptics, NITD has assembled a broad portfolio of early-stage projects by forging research collaborations with academic researchers around the world. “We could never have grown so rapidly if so many people around the world hadn’t been working by themselves on malaria for years and years, without access to pharmacology or drug discovery,” Dr. Matter added. “Now, all of a sudden, you have this marriage of their ideas and our expertise – and things took off.”

Novel Targets

NITD is working with academic partners on compounds with entirely novel targets shown to be essential for survival of the malaria parasite. In one collaboration, NITD is working with Sanjeev Krishna at St. George’s Hospital Medical School, London, to target the hexose transporter in P. falciparum. The hexose transporter controls the supply of glucose to the parasite. Depriving parasites of glucose can kill them within a matter of minutes, significantly faster than artemisinin-based medicines. This class of targets has never been systematically evaluated, however, and the big question is whether such target is “druggable”. In other words, can a medicine be designed that specifically inhibits the activity of the malaria hexose transporter, but also fulfills safety and pharmacokinetic properties required by the development process.

So-called redox enzymes that regulate oxidative status of Plasmodium represent another novel target class that, when inhibited, kills the parasite. But once again, druggability of the target remains in doubt. The redox-enzyme program is a collaboration with Professor Katja Becker at the University of Giessen, Germany. The Genomics Institute of the Novartis Research Foundation (GNF), based in La Jolla, California, is assisting NITD with the high-throughput screening program for redox enzymes.

Additional research projects were reported during poster sessions at the Global Infectious Diseases Workshop in Siena.

Novartis scientists have worked for years on a family of enzymes called aspartyl proteases that are believed to play a role in disorders ranging from oncology to Alzheimer’s disease. Bruno Martoglio, a NIBR biologist and one of the world leading experts in the field, suggested to his NITD colleagues that a relative of the aspartyl proteases – the signal peptide peptidase in the malaria parasite – might be an attractive target.

To succeed, the program must develop lead compounds with selective activity against the malaria version of the enzyme, and at the same time demonstrate that interfering with its function can quickly kill the parasite.

Meanwhile, NITD is also taking advantage of research initiated at GNF in recent years, aiming to increase understanding of the molecular basis of malaria to facilitate drug discovery.

The full sequence of the P. falciparum genome has been available since 2002. The function of most of the parasite’s genes remains uncharacterized, however, particularly genes involved in life-cycle phases such as liver invasion and interactions between the parasite and host.

“These actually may be the best targets – so there is a huge amount of basic biology we need to do,” said Elizabeth Winzeler, a Program Director in Cell Biology at GNF, as well as Associate Professor in Cell Biology at Scripps Research Institute that, like GNF, is based in La Jolla, California. (Click here to learn more about GNF’s research into the molecular basis of malaria)

Objectives of the NITD malaria program, agreed with the Wellcome Trust and MMV, call for two compounds to complete the compound selection process (CSP) by 2009. In Novartis research parlance, CSP is the stage of drug discovery following lead optimization, but prior to initial proof-of-concept studies in humans. Dr. Matter is upbeat about the current portfolio but after decades of industry experience, he has no illusions about the odds against success.

“You need a bundle of things because you can be absolutely sure that 80% of what you do is going to fail,” he said. “This early stage portfolio has been subjected to rigorous criteria but the evolution will be very dynamic. You start and stop, it goes up and down, and I’m sure that it will look quite different from today by the end of this year.”

GNF: Basic Biology of the Parasite Life Cycle

Scientists at the Genomics Institute of the Novartis Reserch Foundation (GNF) are breaking new ground in malaria research, applying high-throughput tools to decipher the role of genes through the life cycle of malaria parasites. The approach promises major benefits for NITD’s program against P. vivax, the parasite strain that causes most malaria in Asia and the Americas.

P. vivax is extremely difficult to culture in the laboratory. Moreover, there are major differences in the life cycle of P. vivax and its parasite cousin P. falciparum. For example, after infecting a host some P. vivax parasites don’t develop immediately and can remain in the host’s liver, in a dormant state, for months. “We don’t know anything about this process in the liver – or about interactions between dormant P. vivax parasites and host cells. It’s all just a huge black box,” said Elizabeth Winzeler, Program Director in Cell Biology at GNF.

“The approach we’re using is to collect detailed gene expression data from many different stages of the parasite life cycle. Then we use bioinformatics to create models for what many of the uncharacterized genes are likely to be doing,” said Elizabeth Winzeler

“The approach we’re using is to collect detailed gene expression data from many different stages of the parasite life cycle. Then we use bioinformatics to create models for what many of the uncharacterized genes are likely to be doing,” Dr. Winzeler added.

Analysis of sequence data for P. falciparum indicates that more than 4,000 genes are transcribed and translated into proteins at one or more stages of the parasite’s life cycle. While some lab tools for dissecting function of unknown genes are available, most such tools have low efficiency and are time consuming, underscoring the need for development of new, high-throughput techniques.

In a landmark experiment in 2003, scientists at GNF and Scripps mined the P. falciparum genome sequence to identify 500,000 short sequences within genes. Those sequences were incorporated into gene chips which were used to determine the relative level and temporal pattern of expression of more than 95% of P. falciparum genes as the parasite moves through its life cycle. Data collected during the experiment provided clues about the potential cellular roles of more than 1,000 hypothetical proteins.

One particular focus, for example, is the stage when parasites emerge from an infected patient’s liver as so-called merozoites and invade red blood cells in the bloodstream, where they feed and multiply further. “At this point, importantly, there is no immune response from the host,” Dr. Winzeler mused. “There probably is a lot of signaling going on between the parasite and the host, to dampen the immune response.”

Several genes have been identified as essential for liver-stage function; if the gene is knocked out in test-tube models, the parasites no longer develop in the liver. “You can imagine if you had a small molecule that would target this protein, encoded by an essential gene, you possibly would be able to knock out liver stage infection as well as increase immunity to the parasites,” Dr. Winzeler said.
“ We have a lot of additional proteins that show very similar expression profiles and are likely to be involved in liver stage function. And some of them are potentially druggable.”

Genome mining also pinpointed a family of kinases found in parasites and in plants – but not in humans. “We became interested in this kinase as a potential target because it is expressed at pretty high levels at the point where the parasite breaks out of red blood cells,” Dr. Winzeler said. “And we have expertise in kinase biology at GNF, of course, as well as a legacy of antikinase drug discovery at Novartis.”