-121-
5 Interaction of approved drugs with P2X receptors
-122-
The successful repositioning of drugs needs careful considerations in order to enhance the probability of matching for various medical problems. The process can be facilitated considerably by application of computational methods e.g. biology, chemo- and bioinformatics in order to combine the information about known targets, drugs, disease biomarkers and pathways (see Figure 5.2). The starting point for a drug repositioning study can be a drug itself, a certain target or a specific disease. The method of choice depends on the starting point and the available information.
Figure 5.2: Flowchart demonstrating existing drug repositioning methods.276
In a target-based drug repositioning approach, the affinity and/or activity of drug library compounds at a known target structure like a protein (e.g. receptor or enzyme) or a biomarker is determined via actual screening (HTS) or virtual docking experiments. A successfully validated assay system or a good virtual target fit allows fast screening of large compound libraries.
Depending on the selected method, valuable hits with minimal effort and low cost can be identified. A disadvantage is that high potency at the target structure may not automatically be relevant for disease pathogenesis. It may also not be sufficient enough to be applied as therapy.
Since the target-based drug repositioning approach does not render much information about new mechanisms beyond the known target, a method using knowledge is much more useful for predicting e. g. unknown drug targets, drug-drug similarities, interactions or new disease biomarkers. Specific genetic signatures are further aspects considered in computational methods for drug repurposing. These signatures include any genetic altering responsible for
-123-
complicated metabolic or signaling pathways. The inclusion of genetic signatures enables the connection of different disease alterations and known protein interactions for better characterization of the mechanism of action of drugs.276-278
It is reported that out of the 75 first-in-class drugs approved between 1999 and 2008, containing 50 small molecules (67 %) and 25 biologics (33 %), 28 of the small molecule drugs were discovered by phenotypic drug screening and 17 by target-based methods. Both methods do not require pharmaceutical or biological information, and can easily be applied to a large number of drugs and diseases.279 Phenotypic screening is the oldest and most important method applied for the search of new and rediscovering drugs. It often starts with clinical observations rendering a certain hitherto unknown pharmacological effect. The general alteration of an entire phenotype to a desired state and knowledge about the mechanistic insight of a specific drug are very useful tools in the discovery of first-in-class drugs for unknown disease patterns and orphan diseases.280-283
The use of large publically accessible drug libraries like the National Center for Advancing Translational Sciences (NCATS) or the Open Innovation Drug Discovery program (OIDD) at Lilly is a useful tool for strategical organization of drug repurposing.273, 283-286
NCATS contains the largest public repository of approved drugs including those in clinical trials. Both allow access to large small molecule libraries directly applicable to in vitro screening assays for fast and effective determination of potency. A library of 600 compounds from NCATS was recently used in a study to identify potential drugs capable of inhibiting the entry of Ebola virus-like particles in HeLa cells. The screening identified 53 hits.287 Another screening experiment of 2600 FDA-approved drugs to find anti-Ebola-active compounds rendered 80 Ebola inhibitors, with matching hits like selective estrogen receptor modulator toremifene, antihistamines bentropine and clemastine, tricyclic and tetracyclic antidepressants clomipramine and maprotiline and microtubule inhibitor vinorelbine.287, 288
Drug repurposing is preferably used for discovery of new agents for orphan, particularly dangerous and acute diseases lacking proper and effective drugs. Efforts to discover new cancer therapies are also exceptionally strong. For example, next to their capability of blocking Ebola virus entry into cells, tricyclic antidepressants like imipramine and promethazine were identified to be potent growth inhibitors of small cell lung cancer and other neuroendocrine tumors via inhibition of caspase 3, among other targets.289 Ion channels proven to be involved in oncogenesis, like potassium and voltage-gated calcium channels, were recently the focus of a study investigating the anticancer effect of approved drugs on these targets.275
-124-
Another very prominent example of successful drug repurposing is sildenafil, a PDE-5 inhibitor.
Originally designed as an antihypertensive drug, the blood pressure-lowering effect in early studies was proven to be only moderate. Simultaneously, the positive effect on penile erectile dysfunction due to the involvement of PDE-5 in the incurrence of an erection was discovered, and clinical studies with a new outcome were designed, leading to the approvement of sildenafil (Viagra®) as the first orally available drug to treat penile erectile dysfunction in 1998 by Pfizer Inc., New York, NY, USA. Since then, due to the strong and selective inhibition of PDE-5, sildenafil has been approved for the treatment of pulmonary hypertension (Revatio®) by the FDA.290 The drug is not recommended for children in this indication.291
Another very prominent example for successful drug repositioning is thalidomide. It was originally approved as a sedative in 1957 under the tradename Contergan® by German company Grünenthal, with recommended use particularly for pregnant women.292 This indication was based on the lack of toxicity in rodents when used in high doses.293 The discovery of the direct association between the intake of thalidomide during early pregnancy and infantile congenital abnormalities led to the retraction of the drug from the market and to a complete change of procedure of therapeutic drug approval.294, 295 However, in the past years, thalidomide was rediscovered as an active and potent agent against multiple myeloma in combination with dexamethasone296 and against erythema nodosum leprosum. The latter was already discovered in 1965.297 In Germany it has been available since 2009 only by submission of the so-called T-prescription. It is part of the T register, which supervises the prescription and emission of thalidomide and its derivatives lenalidomide and pomalidomide.298, 299 Both derivatives are also approved for the treatment of multiple myeloma. The strict control of prescription serves the prevention of deformities in newborn children. Strict safety measures were demanded from all EU member states by the European Commission.
Since the rediscovering of approved drugs for new indications, diseases and targets can be a successful strategy, we tested a small library of 440 drugs for inhibitory potency at purinergic P2X receptors. The aim of this study was the discovery of new potent and selective ligands for various P2X subtypes. The library was screened at each receptor subtype expressed in 1321N1 astrocytoma cells in a final concentration of 20 µM via measurement of calcium influx.
Subsequently, concentration-response curves were generated for active compounds. The results are discussed in the following chapter.
-125-