Pharmacological Profiling of Novel PP-Inhibitors Derived from Cholenic Acid for Targeting Eph-Ephrin Signaling in Glioblastoma

There are mounting evidence that the Eph-ephrin system is involved in several pathological processes, especially in cancer. Altered expression of this system has been observed in many tumours such as breast, colon, liver, prostate, and glioblastoma. Eph receptors and their ligands have been related with malignant progression, tumour angiogenesis, metastasis, propagation and maintenance of tumour stem cell. Looking at this evidence, it is reasonable the idea to target this system as a new or alternative therapeutic strategy in oncology. Different approaches to interfere with Eph/ephrin signalling have been explored over the years and some of them have reached clinical trials, showing contradictory results. Since 2009, our research group has focused its attention on the development of small molecules able to hamper the Eph-ephrin binding by targeting the extracellular ligand binding domain of Eph receptor. As noted in the introduction, UniPR1331, an Eph receptors pan-inhibitor, has demonstrated activity in disrupting the EphA2-ephrin-A1 interaction and has shown efficacy in reducing tumour growth in an in vivo model103. However, it is important to remember that the Eph-ephrin system is a redundant system, in which almost every ephrin can bind to multiple Eph kinases67. Thus, the silencing of one single ephrin-or Eph kinase-could easily be compensated by other members of the family. Thereby, this redundancy makes it difficult to assess to what extent the two subfamilies, A and B, each contribute to the various processes that support tumor growth. Thus, my research might be the starting point for a more extended investigation that will contribute to explaining the role of these two subfamilies in detail in different tumorigenic processes. Indeed, the treatment with selective molecules active on either A or B subfamily could clarify the role played by the two subfamilies in modulating the different tumorigenic processes of glioblastoma. Based on that and thanks to the computational analyses developed by Professor A. Lodola’s research group, the synthesis of UniPR1447 was achieved. UniPR1447 is a beta-homologue of UniPR1331. UniPR1447 (1), specifically, is the L-β-homotryptophan conjugate of 3-β-hydroxy-Δ5-cholenic acid. This compound interacts with the EphA2 receptor by accommodating its rigid core within a hydrophobic channel, with its carboxylic group forming a salt bridge with Arg103 and its indole ring positioned in an accessory pocket adjacent to Met73. The presence of this accessory pocket is a distinctive feature of the ligand-binding domain (LBD) of EphA2, absent in EphB2. Exploiting this unique characteristic, a N-sulfonylphenyl substituent was introduced at the indole nitrogen atom of UniPR1447(1). This modification led to the synthesis of compounds UniPR1449 (2). These compounds exhibited significant affinity for the EphA2 receptor while demonstrating no interaction with different EphB receptors at concentrations up to 30 μM. The introduction of the bulky N-sulfonylphenyl substituent conferred a degree of selectivity for EphA2, underscoring the potential for developing EphA2-specific antagonists.UniPR1331 derivates were pharmacologically characterized with the aim to identify potent Eph binders, interfering with the Eph/ephrin system and endowed by anti-tumoral properties. As first step, all new synthetized molecules have been tested in displacement studies in order to select only the most potent. Then, these selected compounds were investigated for their ability to inhibit the EphA2 activation, cytotoxic effects and through functional assays performed on cells. Finally, compounds were tested in pharmacokinetic studies.