Re-Pharm secures patent on RP0217, a re-purposed drug discovered using Cresset’s Forge

The discovery of novel biology elucidated the mechanism of action of a series of old anti-inflammatory compounds. Re-Pharm scientists used Cresset’s software to find an active compound that Re-Pharm has patented as a novel anti-inflammatory agent with ‘steroid sparing’ capability.

The discovery and use of chromones as anti-histamines

In the 1950s Dr Roger Altounyan of Fisons Pharmaceuticals, researched the traditional Egyptian herb Khella as a potential treatment for asthma. He identified the compound Khellin and various derivatives including a series of compounds known as chromones after the key chemical structure scaffold. Chromones are effective in preventing the release of histamine and they include sodium cromoglycate (cromolyn), which is still widely used today. Altounyan and his co-workers identified these compounds as ‘mast cell stabilizers’, but they were not able to elucidate their underlying mechanism of action.

The role of Annexin A1 in the inflammatory response

Annexins are a family of small proteins which play a number of roles within the body. Annexin A1, a 40 kDa protein formerly known as Lipocortin 1, has a role in mediating the inflammatory response. It is a potent inhibitor of Phospholipase A2 and thereby inhibits the production of key pro-inflammatory agents such as eicosanoids and leukotrienes. Annexin A1 is activated and released from various cells in response to binding of endogenous or synthetic glucocorticosteroids to the glucocorticoid receptor.

Professor Rod Flower and his group at the William Harvey Research Institute discovered that human Annexin A1 mediates the inflammatory response in various animal models of inflammatory diseases. They found that the mechanism of action of steroid drugs, acting via Annexin A1, works through phosphorylation of intracellular Annexin A1, which is then localised to the cell membrane and excreted. It is the phosphorylated Annexin A1, rather than the direct action of the steroids, that shows the strong anti-inflammatory response. Dephosphorylation of activated Annexin A1 leads to its deactivation and the cessation of the downstream cascade of anti-inflammatory effects.

Professor Flower and colleagues found that the chromones block the de-phosphorylation of Annexin A1, thus extending its anti-inflammatory action. They had finally identified the chromones’ elusive mechanism of action. Furthermore, they were also able to identify the enzyme target Protein Phosphatase 2A (PP2A) as key to turning off the action.

A new protein target for anti-inflammatories

PP2A represented a new target for anti-inflammatories. Chromones are active against PP2A but they have very poor oral pharmacokinetics. As a result they are mainly used topically. The identification of new compounds that are directly active against PP2A has the potential to provide an alternative to steroids.

Re-Pharm is a UK-based company that specialises in the re-profiling of existing drugs. They set out to identify an existing compound that was also active against PP2A that could be re-purposed as an anti-inflammatory.

The re-profiling of existing drugs for new disease indications has a number of advantages, all based around the principle that there is existing knowledge about the candidate compounds. This leads to shorter development cycles and in some cases faster regulatory approval.

Cresset software used to identify new active structures

The starting point for Re-Pharm’s search was the three ligands known to be active at PP2A, plus a crystal structure of PP2A bound to a large toxin that completely shut off its mechanism. Based on these, Re-Pharm used Cresset’s computational chemistry software to build a template for a compound that would be active at PP2A.

Chemical structures of the three chromones known to be active at PP2A
Figure 1: The chemical structures of the three chromones known to be active at PP2A: nedocromil, cromolyn and the progenitor compound khellin.

The Re-Pharm scientists used the molecular modeling software Forge from Cresset to generate proposed bioactive conformations for each of the molecules. Re-Pharm used Forge to create field templates of three of the chromones that were now known to be active at PP2A. Each proposed bioactive conformer with its Cresset field point patterns was screened separately against a database of known drugs to find compounds with similar activity.

The crystal structure was also imported into Forge and the bound ligand was manually trimmed back in order to identify the active pocket in the protein. This was used to create a fourth template for a screening run.

The four hit lists were fused to deliver an overall set of known drugs which had the potential to be active against PP2A.

Screening results in patented compound

The results of the four runs were evaluated and prioritised. A small number of compounds were purchased and screened. Within the screened compounds, one was found to inhibit PP2A with nanomolar activity. This existing compound is known internally as RP0217.

The initial hit was followed up with secondary screening, including ex vivo and in vivo models of inflammatory diseases. Through all of these validation experiments, RP0217 has been found to be an effective anti-inflammatory agent, and has consequently been patented by Re-Pharm for a variety of disease indications.