Development of a high-content intracellular signalling pathway analysis methodology for drug repurposing

It is estimated to cost several billion pounds to get a new drug to market and many fail in development, despite showing promising effects. Therefore, "repurposing" of an existing drug to a "new indication" (i.e. applied to a new disease) offers significant cost and efficiency benefits to pharmaceutical companies in the search for new marketable drugs. Prior work on drug delivery, production and safety can be re-utilized and time to market is greatly reduced. Prime examples are Thalidomide, now successfully used in treating both leprosy and multiple myeloma, and Viagra to treat the rare disease pulmonary arterial hypertension and more recently altitude sickness. Systematic repurposing requires identifying the effects of a drug upon proteins ("targets") within a cell, which change the behaviour of the cell. With sufficient knowledge of the range of targets that are altered in a particular disease, one can screen for drugs that inhibit the disease related effects. We have developed a methodology that can measure simultaneously many target proteins in large numbers of samples. These targets can be proteins involved in controlling the behaviour of cells, eg. signalling and regulatory proteins. We are confident that this methodology can be applied to screen large numbers of existing drugs for repurposing and we propose to demonstrate this in a disease in which we already have a great deal of research experience and expertise: this is a rare genetic inflammatory disease, TNF Receptor-Associated Periodic fever Syndrome (TRAPS).
Rare diseases (including TRAPS) are defined as those that affect less than 1:200,000 of the population: it is therefore simply not cost-effective to develop drugs specifically for these conditions. Thus, most drugs used to treat rare diseases are the result of repurposing of existing drugs. Identification of disease-related, protein targets for rare diseases (disease profiles or "signatures"), combined with systematic screening of drugs for repurposing, offers a great hope for identifying new treatments from the many thousands of existing drugs.
TRAPS is the result of mutations in a cell-surface receptor (TNFR1), usually involved in inflammatory responses to infection. TRAPS patients are hypersensitive to inflammatory stimuli and suffer from debilitating bouts of fever and systemic inflammation. We have worked for many years to understand the cellular mechanisms of the syndrome and have identified a signature of over 20 proteins within cells that are associated with abnormal inflammatory signalling in TRAPS.

We will automate our existing technology so that it can be used as a drug repurposing tool. As such, it will support the mass exploration of alterations in proteins within cells after exposure to potential drugs. Automation would make it possible to expose cells to many thousands of drugs and enable each exposed cell culture to be examined for at least 40, and potentially many more, targets.
This development requires changes to our methods to enable robotic high-throughput processing and ensure appropriate reproducibility and quality control. We will test the system's sensitivity and operational robustness. We will finally use our established TRAPS model as a test-case and screen a library of 1440 established drugs for their effects upon our TRAPS protein signature (measuring 40 targets for each drug tested). Identified positive "hits" will be subsequently investigated for their benefit in TRAPS.

Our platform for drug repurposing will be widely applicable wherever in-depth analysis of complex cellular behaviour can facilitate drug development, including cancer, infectious diseases, autoimmunity, allergy, and the multitude of rare diseases lacking effective drugs. We believe this approach offers direct benefits to large and small pharmaceutical companies, healthcare providers and academic groups alike, who need to comprehensively examine multiple protein targets for many samples.

We have developed a high-content protein analysis pipeline with high throughput potential, enabling components (50+) of multiple signaling pathways to be monitored simultaneously within large sets of biological samples. We will develop the methodology's potential as a drug repurposing tool for automated mid to high-throughput use.
Repurposing of existing drugs for use in other diseases represents a highly cost effective route for pharmaceutical companies to gain new market opportunities for existing drugs and for re-examining failed compounds which showed promising characteristics. It also represents an important route to discovery of new therapeutics for a multitude of rare diseases, which are not economically attractive for major research and development by pharmaceutical companies.
As a test case we will develop our platform to evaluate a compound set, consisting of marketed and off-patent drugs and known pharmacologically active compounds, for effects upon the defined intracellular signaling signatures which we have determined in the orphan autoinflammatory disease, TRAPS (TNF receptor-associated periodic syndrome). We have detailed knowledge of the signalling abnormalities caused by the presence of mutated TNF receptor 1 (TNFR1) in this rare syndrome. TRAPS associated mutations perturb the baseline balance of intracellular proinflammatory signals, resulting in hyper-responsiveness to encountered inflammatory triggers. No satisfactory small molecule drugs are available. Adequate treatment will therefore be best met by drugs that can restore signaling homeostasis. Screening for such drugs necessitates a multiplexed analysis of the perturbed pathways.
Our platform will provide high-throughput, multiplexed reporting across a biomarker "landscape" of over 40 protein targets, suitable to such goals. It will provide a powerful, integrated system for re-examination and repurposing of drugs applicable to both orphan and common disease indications.

Repurposing existing compounds for new indications offers great potential to maximize a compound's usefulness, by utilizing existing pharmacological and clinical data to support the new application whilst minimizing development cost, lead-time and accelerating time-to-market. A large-scale, high-throughput analysis of multiple (50+) markers (signaling intermediates, adhesion molecules, transcription factors etc), would provide a fine detail map of a compound's effects across interdependent pathways, offering a unique approach applicable to drug reprofiling.
Our proposed repurposing platform would offer this capability, scalable to allow thousands of compound exposure experiments, with each exposed cell culture being profiled for many target molecules. This represents a tremendous opportunity to simultaneously examine both on and off-target effects of a drug upon cellular behaviour

WHO WILL BENEFIT FROM THIS RESEARCH AND HOW?
- Large Pharmaceutical companies, SMEs and academic groups who have bioactive compounds, or other agents (siRNA, biologics), and require multiplexed analysis of many intracellular pathways, will benefit by access to the platform. Large scale, massively multiplexed analysis of intracellular signalling events will support better understanding of both on and off-target actions upon target cells and systems.

- Systems biology and mathematical researchers developing models of cellular behaviour and drug effects upon cellular pathways will benefit through the availability of content-rich, numerically quantified datasets to support improved mathematical models of dynamic cellular processes and drug effects.

- Translation medicine researchers, and research involving preclincal and clinical trials will benefit where comprehensive protein profile monitoring in large patient cohorts is required during and after drug treatment. Such research will also benefit through improved capabilites for initial marker profiling to identify novel disease signatures. This has immediate application across common inflammatory conditions (RA, SLE), cancer, infectious diseases and allergy.

- International collaborations will benefit. Our initial signalling methodology has attracted considerable attention from national and international research groups interested in collaborating with us. A working repurposing platform would support straightforward collaborative access and allow many groups to benefit from applying the technology within their own field.

- Healthcare providers will benefit from improved choice of (repurposed) drugs to combat difficult disease indications. This has significant cost-saving implications, for instance where a specific small drug therapy can replace expensive biologics.

- TRAPS sufferers, and more widely those many millions of people affected by rare diseases worldwide will benefit by improved prospects of affordable drug-screening being applied to identify existing compounds with beneficial characteristics in such rare diseases.

- Animal Health researchers will benefit from adaptations of the technology to both large and small animal species, providing benefits similar to those outlined for human studies.

- Work within the remit of the the NC3Rs (replacement, refinement and reduction of animal use in research) will benefit from adaptation of the platform for use in mouse and other experimental animal models. Researchers can maximize intracellular and extracellular data obtained from each animal studied, with potentially every organ being biopsied, reducing overall animal usage.