LONDON—X-ray crystallization is a technique that enables scientists to get an idea of the structure of a crystal on a molecular and atomic level, and is particularly helpful in visualizing receptors and molecules in drug discovery to discern potential binding sites. Using this technique for just that purpose, Heptares Therapeutics, the wholly owned subsidiary of Sosei Group Corp., recently announced the first resolved high-resolution X-ray crystal structures of the protease-activated receptor 2 (PAR2) together with antagonist molecules. The paper, “Structural insight into allosteric modulation of protease-activated receptor 2,” appeared online in Nature.
PAR2 is a G protein-coupled receptor (GPCR) known to be a target for many indications, and is activated by cleavage with a protease enzyme in a way that the cleaved part of the receptor serves as its own ligand. Despite being well validated, it has been markedly difficult to target PAR2 using traditional drug discovery tactics given its abnormal nature.
“PAR2 is an important target that is linked to multiple disease areas (e.g. pain, cancer and inflammatory diseases), but which has previously proved intractable to conventional drug discovery approaches,” says Fiona Marshall, co-founder of Heptares and chief scientific officer of Heptares and Sosei. In fact, she notes, “The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. There are no drugs so far which target PAR2.”
The findings featured in the Nature paper are the result of a long-term collaboration between Heptares and AstraZeneca scientists to discover novel PAR2 antagonists, which were then crystallized in complex with the PAR2 receptor as well as having their X-ray structures described with high resolution.
In particular, the results focus on AZ8838, a small-molecule antagonist identified by AstraZeneca via high-throughput screening. It was found to bind to PAR2 in a fully occluded and previously inaccessible pocket near the receptor’s extracellular surface. In addition, another molecule, AZ3451, was identified by screening compounds with the PAR2-StaR protein and was shown to bind to a remote allosteric site believed to prevent structural rearrangement necessary for receptor activation and signaling. A blocking antibody fragment was also found to bind to PAR2’s extracellular surface and prevent the ligand’s access to the binding site.
Niek Dekker, principal discovery scientist of innovative medicines and early development at AstraZeneca, commented: “Access to the Heptares StaR technology enabled us to progress available small-molecule actives on PAR2 to credible lead series, where we earlier struggled to develop our chemistry. We are particularly excited by the functional and binding study data from one of the lead series as this exhibits slow binding kinetics, which is an attractive feature for this target.”
“Heptares’ technology was used to stabilize the PAR2 receptor and allow its crystallization bound to several antagonist molecules, including small molecules and antibodies. These crystals could then be analyzed using high-power X-ray to determine the structures—this has never been done before and has generated important structural information about the receptor and how it interacts with antagonists, which will be crucial to the optimization of antagonist molecules for further development,” says Marshall. “The purified PAR2 StaR protein was also used for high-throughput screening at X-Chem, which enabled the discovery of novel antagonist molecules that were subsequently crystallized with the PAR2 receptor. Given the importance of PAR2 and the previous lack of success in targeting it, this is a great step forward.”
A Heptares StaR (stabilized receptor) protein “is a GPCR with a small number of point mutations that greatly improve its thermostability without disrupting its pharmacology. StaR technology is transferrable across the GPCR superfamily and allows the selection of stable, functionally relevant, purified conformations of target GPCRs that retain their expected drug-binding characteristics. Whereas unstable wild-type proteins are intractable to structural studies, StaR proteins allow structure determination and provide the launch pad for an SBDD approach to GPCR drug targets,” the company explains on its website.
Marshall says that work on this project will continue at AstraZeneca, applying both “the X-ray structure and [structure-based drug discovery] methods to optimize lead compounds towards candidates.”
She also tells DDNews that Heptares and AstraZeneca have worked together on several targets in a number of therapy areas of interest to AstraZeneca since 2011, and agreements were signed in 2015 for the development of a novel adenosine A2A receptor antagonist discovered by Heptares. That candidate, AZD4635, is now in Phase 1 trials in solid tumors as a single agent and in combination with AstraZeneca’s durvalumab, she adds.
Heptares announced back on April 5 that it had reached a milestone in its collaboration with AstraZeneca on AZD4635, one that triggered a payment of $12 million from AstraZeneca. The milestone was reached with the completion of a preclinical program that showed the compound’s effect in reversing adenosine-mediated T cell suppression and boosting antitumor immunity. AZD4635’s ability to block A2A signaling demonstrated the capacity to reduce tumor growth both when administered alone and in combination with anti-PD-L1 checkpoint inhibitors. The two companies presented their results at the 2017 American Association of Cancer Research annual meeting.