Design and synthesis of new PPI inhibitors of the EphA2 receptor targeting Glioblastoma Multiforme (GBM)

Compelling evidence shows that the Eph-ephrin system has a cardinal role in embryonic development, with minimal implications in the physiology of the adult. However, Eph receptors – in particular the EphA2 receptor subtype – have been found often overexpressed and deregulated in several tumours including Glioblastoma Multiforme (GBM), with a primary involvement in tumorigenesis, angiogenesis, and cancer stem cell regeneration. Preventing EphA2 receptor activation by ephrin-A1 with small molecules may provide new agents useful in the treatment of GBM, as shown by the recent finding reported for UniPR1331, the L-Trp conjugate of 3β-hydroxy-Δ5-cholenic acid. Despite its promising activity in both xenograft and orthotopic mice models of GBM, UniPR1331 suffers from suboptimal potency, lack of selectivity and rapid degradation after oral administration. In this Ph.D. thesis, two new classes of amino acid conjugates of 3β-hydroxy-Δ5-cholenic acid derivatives were designed, synthesized and evaluated as antagonists of the EphA2-ephrinA1 interaction with the aims of identifying new EphA2 antagonists featuring higher potency and selectivity. Selectivity has been achieved targeting a peripheral pocket of the EphA2 ligand binding domain, not present in the EphB receptor subfamily, by the selective functionalization of indole nitrogen of the L-β-homotryptophan moiety conjugated to the 3β-hydroxy-Δ5-cholenic acid. Among the first class of UniPR1331 analogues synthesized, UniPR1454 (N-(3β-hydroxy-Δ5-cholen-24-oyl)-(4-(trifluoromethyl)phenyl)sulfonyl)-L-β-homotryptophan) emerged as a fair compound, displaying low micromolar affinity for EphA2, improved selectivity over EphB receptors and significant antiproliferative activity in GBM cell lines. A second class of UniPR1331 derivatives was synthesized introducing a diverse set of 3β-carbamoyloxy substituents on the Δ5-cholenic acid scaffold. A SAR exploration showed that small lipophilic substituents at the carbamate nitrogen atom not only were tolerated by the EphA2 receptor but ensured a 10-fold increment in the affinity. UniPR1479 (N-(3β-(ethyl-carbamoyl)oxy-Δ5-cholen-24-oyl)-L-tryptophan) displayed an inhibitory constant (Ki), relative to the disruption of the EphA2-ephrin-A1 complex, in the sub-micromolar range and promising in vitro antiproliferative inhibition on GBM cell lines. A future in vivo evaluation of these new compounds will allow to assess their ability to act as anti-GBM agents.