The EphA2 receptor and its ephrin-A1 ligand form a key cell communication system, which has been found overexpressed in many cancer types and involved in tumor growth. Recent medicinal chemistry efforts have identified bile acid derivatives as low micromolar binders of the EphA2 receptor. However, these compounds suffer from poor physicochemical properties, hampering their use in vivo. The identification of compounds able to disrupt the EphA2-ephrin-A1 complex lacking the bile acid scaffold may lead to new pharmacological tools suitable for in vivo studies. To identify the most promising virtual screening (VS) protocol aimed at finding novel EphA2 antagonists, we investigated the ability of both ligand-based and structure-based approaches to retrieve known EphA2 antagonists from libraries of decoys with similar molecular properties. While ligand-based VSs were conducted using UniPR129 and ephrin-A1 ligand as reference structures, structure-based VSs were performed with Glide, using the X-ray structure of the EphA2 receptor/ephrin-A1 complex. A comparison of enrichment factors showed that ligand-based approaches outperformed the structure-based ones, suggesting ligand-based methods using the G-H loop of ephrin-A1 ligand as template as the most promising protocols to search for novel EphA2 antagonists.
The Eph receptor-ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The l-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small-molecule antagonists of the EphA2 receptor.
Heparanase is the sole mammalian enzyme capable of cleaving glycosaminoglycan heparan sulfate side chains of heparan sulfate proteoglycans. Its altered activity is intimately associated with tumor growth, angiogenesis, and metastasis. Thus, its implication in cancer progression makes it an attractive target in anticancer therapy. Herein, we describe the design, synthesis, and biological evaluation of new benzazoles as heparanase inhibitors. Most of the designed derivatives were active at micromolar or submicromolar concentration, and the most promising compounds are fluorinated and/or amino acids derivatives 13a, 14d, and 15 that showed IC50 0.16-0.82 μM. Molecular docking studies were performed to rationalize their interaction with the enzyme catalytic site. Importantly, invasion assay confirmed the antimetastatic potential of compounds 14d and 15. Consistently with its ability to inhibit heparanase, compound 15 proved to decrease expression of genes encoding for proangiogenic factors such as MMP-9, VEGF, and FGFs in tumor cells.
The Eph-ephrin system, including the EphA2 receptor and the ephrinA1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist that is able to inhibit EphA2 receptor activation; it is therefore potentially useful as a novel EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of lithocholic acid has a negligible role in recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand binding domain of EphA2, with a steady-state dissociation constant (K(D) ) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1 ligand. These findings suggest that cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.
Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.
