Collaborating to find new approaches to tropical disease

There has been a sea change in the way many biotech and pharma companies view the search for new drugs in neglected disease. Serono is a biotech company, with interests in neurology, reproductive health, oncology and dermatology – but we teamed up with the World Health Organization (WHO) to train two visitors in finding new medicines. From this simple example, we have learned much that shows that beyond simple corporate social responsibility we have much to benefit from with such collaborations.

There is a great need for new medicines for treating tropical disease. Even today, tropical parasitic diseases such as Malaria, Leishmaniasis, African sleeping sickness or Chagas disease still represent a major health threat for a majority of the world population. They lead to many millions of deaths per year, and massive socio-economic effects in many countries. New drugs should be focused on the needs of target patients, and be suitable for use in the field. They need to be cost-effective, orally bio-available and stable under extreme conditions of heat and humidity.

Serono is Europe’s leading biotechnology company, with products in reproductive health, neurology, psoriasis and immunology, headquartered in Geneva. We have no ongoing research programs in the area of Tropical Disease. In 2004 we started a project in collaboration with the World Health Organization to train two visitors in drug discovery from our perspectives, using targets from tropical disease. We screened two targets against our compound collection and identified interesting starting points for medicinal chemistry projects. – At the same time, we were able to train visitors in the process and technologies of drug discovery. From Serono’s perspective, the project has enabled us to screen target classes, which are new for us, and identify new chemotypes of inhibitor. More important than that, has been the wide network of collaborations that came from the project, and that our work with the WHO has become an important part of our social responsibility as a drug discovery organisation.

There are many possible ways that the biotech industry can form partnerships to become involved in developing drugs for neglected disease1. For Serono, one of the key points is that we do not have any ongoing programs in infectious disease – our in-house focus being on developing life changing treatments for non-communicable diseases. This made the collaboration with the WHO much simpler in many ways, since there was no possibility of a conflict with in-house anti-infective programs. The project was a success because of the involvement of so many people within our organisation who gave freely of their spare time to advise, coach and challenge the project. It started with a commitment to screen against molecular defined targets involved in two tropical diseases, malaria and leishmania. Any compounds that were shown to be active (at sub-micromolar concentrations), selective, novel and non-toxic were considered hits. These could then be tested in more detailed cell biology in collaboration with the WHO and academic partners. This is a process, which is going on as we speak.

We invited two exchange visitors to work with us for 12 months to set up these screens, and to learn about our view on screening. Our strategy for screening has always focused on high content screening and high quality assays rather than on ultra high throughput only2. High content, information rich, means looking for as many possible descriptors that show your compound has the desired effect in a system as close to physiological reality as possible. Following our strategy, our robotics and automation systems are centered around 96 and 384-well systems. Assays which were chosen are therefore easy to transfer to external sites, and we fundamentally believe that the technology transfer will be fruitful, should we decide in the future to run similar experiments in the laboratories of our exchange visitors (in Brazil and Cameroon).

Targets and compounds to screen

For our first batch of targets, we used ones for which assays had already been set up – and for this we were greatly assisted by our colleagues at the WHO. Despite the recent increase in screening neglected disease targets that we see in many companies, there was still a need for someone to help with the initial contacts. During the two years that we have been involved in this project, we have witnessed a significant lowering of defensive barriers from academics and non-governmental organisations – proving that as always the best way to smooth relations is to have successful projects.

The compounds we tested were the synthetic libraries generated for our other in house projects. Where we know which compounds should inhibit, we have focused on those in the assays – but in this disease area much more than any other it is important to add an aspect of serendipity to the process. Results that are easy to obtain usually represent an incremental step forwards. We had hoped to supplement the process with libraries of purified natural products – but so far this has been difficult to organise. We are hoping that in the future our success so far will help us in our discussions to gain access to such collections. Natural products are synthetically more difficult by an order of magnitude – but often tend to be sufficiently potent to be used directly. Effectively this replaces the bottleneck of medicinal chemistry with that of material supply – but it does offer an approach to moving forwards quickly.

Systems parasitology – new approaches based our expertise in target classes

The completion of the human genome sequence in the late 1990s enabled us to exploit the ‘druggable genome’ more completely3. Put simply, this meant that we have expertise in inhibiting or activating a particular target (G protein coupled receptors, or kinases, are two examples) – and therefore could make a list of human targets which we could reasonably expect to modulate, based on our current compound diversity.

The recent sequencing of many parasite genomes has opened up a new field of anti-infectives research – which we call systems parasitology. We now have the complete genomes of infectious agents such as Plasmodium4 and Trypanosoma5 as well as an enormous database of expressed sequence tags. The availability of this sequence data means that we can prioritise some target types – knowing that we already have had success against this class – and so in principle they are druggable.

Examples here would include kinases (or which several have been shown to be essential for growth of Malaria). Often the parasite kinase has the same name as a mammalian protein (GSK3 being a good example) – but there are still good reasons to suspect it will be possible to find selective inhibitors. Similarly, for Phosphatases, there is an enzyme in the Tuberculosis genome, which is required for survival of the mycobacteria. Many of us have worked at length to find inhibitors for a phosphatase called PTP1b – involved in diabetes, and built up a collection of diverse molecules as potential inhibitors. Some of the compounds that were made for this project are active against the infectious disease phosphatase.

So, using this approach we know it’s likely that we will find inhibitors. Better than that, they should be from a series of compounds, which are already known to be reasonably drug like (they are soluble, stable, etc). One key challenge is selectivity – but this has to be defined at the level of the whole organism – does my molecule stop the key parasite process before I see an effect that is detrimental to the patient? We still have a lot to learn about how to relate molecular selectivity to this functional selectivity.

Pay-back time – parasites leading us into new target classes

The other approach to screening targets is a more long-term investment to finding new medicines in infectious disease, but with an interesting potential payback for our research in other therapeutic areas. This is the approach we chose to take with our collaboration with the WHO. The first screen that we worked on was against the serine protease PfSub1 from Plasmodium falciparum – the malaria parasite6. Classical serine protease inhibition has used the fact that there is an active site serine to generate irreversible inhibitors of the enzyme – largely by rational design based on the substrate. Here, we took the approach of specifically looking for micromolar inhibitors of the enzyme, which are reversible.

The assay used was simple and robust – based on quenched fluorescent substrates, which become more fluorescent when cleaved – and was run in a 384-well format. The robustness of the assays illustrates a Z’ value of 0.69 on the robot and of 0.51 to 0.8 when performed manually (a value of 1.0 would be the perfect assay). We particularly like the idea that the assay is equally good when done by hand – since it makes it transportable to a lab with less automation. After screening more than 30,000 compounds, we found some hits. One of the important differences we see between industry and academia is the quality control. Here, we re-synthesised the compounds and retested them before being sure we had active molecules. They are now being tested at the Swiss Tropical Institute in infection assays involving whole parasites – where we can see that their potency is holding up.

So what is the next step? It has often been said that the difficult part of drug discovery does not start until you have compounds that are active in cell assays. Here, we are making libraries of compounds around our original hits, as part of classical hits to leads progression. However, we can add in two extra twists to the process. First, although the design of the libraries will be done in Geneva by our expert chemists, the construction of the libraries will be done by our long term industrial partners in Bangalore, India – making the most of the low cost environment, and the impressive skill base that exists. Second, although none of our starting points for chemistry are classical serine protease inhibitors, we already know that they have activity against other mammalian serine proteases. We therefore have an effective way to produce new chemotypes, using the parasite target as a way of hopping from one part of chemical space to a new one. The fact that the parasite targets are similar, yet different – a sort of chemical half-way house, enables us to do this.

Two way street: what did we learn from our visitors?

It’s easy to see what came out from the collaboration. Our colleagues at the WHO program for tropical disease research identified two Fellows, who came to Geneva for a year and learned all the processes from setting up assays for screening, through to analysing the results and testing the quality of the molecules. Beyond that we have been able to develop three assays, and complete two of them on the robots successfully. We have some starting points for chemistry, and an ongoing project to develop these hits into molecules, which are drug like. At the same time, it has enabled us to ask questions about some other targets, and begin to collaborate with new groups advising them on how to look for druggable molecules.

However, when considered the other way around – then the benefit has been very large too. More than a simple act of corporate social responsibility, we have seen the effect that this collaboration has had on our people. The images left of a medical doctor from Cameroon, learning the skills of drug discovery, but able to impart his medical knowledge and insight at the heart of the screening organisation – a hot bed of technology, often seen as a long way from the hospital bedside. Or a Brazilian natural products chemist, who taught us to see the world from the perspective of his laboratory back home – still driving the process of discovery, but having to find new ways to do things that we think are standard practice. In the process we have all learned from each other – and found new molecules which one day may be seen to be the starting points for new medicines.

In conclusion, we have given comprehensive training to two WHO fellows, who have been able to develop three assays. Two of the assays were performed in house, one in manual mode and the other in fully automated mode. Even more importantly, these assays are easily transferable to developing countries, allowing additional medium throughput screening campaigns to be performed locally on similar targets using available compound libraries.

From the industry point of view, we observed a high level of motivation among Serono employees to contribute to this project. Furthermore, this type of collaboration may have the added benefit of increasing the chemical diversity of compound collections by targeting exotic (non-mammalian) members of interesting target classes. A follow up program on tropical diseases is planned to start by the beginning of Q3/2006.

References

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