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Content archived on 2024-06-18

Destabilization of the Epithelial Tissue Architecture by Competition with e-cadherin Homo-dimer formation: small molecule-induced disruption of the epithelium integrity and functions

Final Report Summary - DETACH (Destabilization of the Epithelial Tissue Architecture by Competition with e-cadherin Homo-dimer formation: small molecule-induced disruption of the epithelium integrity and functions)

In our laboratory, we focus on the production as well as the structural and functional characterization of large biological macromolecules. In particular, we study members of a large family of cell-cell adhesion proteins called cadherins. Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. The over 100 different family members known to date are phylogenetically classified in several sub-classes, mostly according to their overall molecular architecture. To date, the most studied cadherins are those classified as classical, which are further divided into type-I or type-II depending on selected sequence features. As a result of structural and mutational studies on several classical cadherins such as type-I C-, E- and N-cadherin and type-II cadherin-11, cadherin-8 and MN-cadherin. a strand-exchange mechanism has been clearly identified as the “endpoint” of a highly dynamical homo-dimerization process. This mechanism consists in the opening of the N-terminal adhesion arm and the insertion of the side chain of the conserved tryptophan in position 2 (Trp2) into a highly conserved acceptor pocket in the extracellular domain 1 (EC1) of the partner molecule that protrudes from the surface of the neighboring cell, forming the so called strand-dimer.

Unlike other members of the family of classical cadherins, a detailed structural characterization of P-cadherin (placental cadherin) has not yet been fully obtained. P-cadherin is a prominent member of the classical cadherins subfamily. Originally found to be highly expressed in mouse placenta during pregnancy, the protein was later found not to be expressed in human placenta. Although transient P-cadherin expression can be observed in several types of tissues during development, the protein is permanently found only at the level of cell-cell junctions in adult epithelial tissues, partially co-localizing with E-cadherin. Mutations in the gene encoding for P-cadherin (CDH3) have been found to be associated with hypotrichosis and juvenile macular dystrophy, a rare congenital disease that causes progressive retinal degeneration and leads to early blindness. Several studies have also indicated a clear, albeit often conflicting, role of P-cadherin in different types of cancer, such as in malignant melanoma and breast cancer. In fact, whereas it appears to inhibit cell detachment and metastasis in melanoma patients, P-cadherin expression is considered to be a marker of tumor aggressiveness in oestrogen receptor (ER)/progesterone receptor (PgR)/HER2-triple negative breast cancers, usually representing a poor prognosis factor in the triple negative subgroup of patients. Furthermore, P-cadherin up-regulation is observed in gastric, lung, colorectal and pancreatic cancer patients. Hence, efforts to develop therapeutic agents against P-cadherin have recently intensified. Recently, we have managed to obtain the high resolution crystal structure of the closed form of human P-cadherin-EC1-EC2 at 1.6 Å resolution, the highest resolution ever achieved for a multidomain cadherin fragment. Our structure shows a novel packing arrangement that provides a further snapshot in the yet to be achieved complete description of the highly dynamical cadherin dimerization pathway. In fact, this is the first cadherin to be crystallized and structurally characterized in a closed and monomeric conformation. All previous structural determinations had so far lead to the implicit assumption that at high protein concentrations such as those usually obtained in crystallization experiments, strand-dimers would preferentially form in the crystal, leaving the fundamental question as to how this highly dynamic system would shuttle between two forms characterized by essentially similar free energies, the inactive and the adhesive conformation, still unanswered. The structural comparison of different cadherins in their various activation stages can clearly contribute to the complete elucidation of the molecular bases of the cadherin binding mechanism and homo-specificity and help clarify how the entropic cost of dimerization may be overcome in a system where the closed and the adhesive forms of the protein have essentially similar free energies. The P-cadherin structure and packing arrangement obtained in our laboratory provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway and shed light on important issues such as cadherin homophilic specificity and dimerization mechanism.
The variety of transient yet critical key intermolecular interactions involving different residues at different stages of the full cadherin dimerization trajectory play all a critical role in guiding the system through a number of intermediate intermolecular protein-protein arrangements during the recognition process that leads from monomeric cadherin to strand-dimer-formation and back. As a result of this highly dynamic behaviour and despite a growing interest in the field, the rational design of small ligands targeting cadherins protein-protein interactions is still in a very early stage. It is worth stressing that targeting the interfaces between proteins has huge therapeutic potential, but to discover small drug-like molecules that are capable of modulating those protein-protein interactions that are by their very nature highly dynamical is still a challenge. In fact, it is clear that the dynamic features of the cadherin dimerization process are likely to play against a traditional drug design approach. Accordingly, the design of a stable molecular interactor against the adhesion pocket would not just prove difficult but it would fail to achieve complete inhibition. However, peptidomimetic molecules that can transiently interfere with structurally validated intermediates in the cadherin dimerization pathway may successfully modulate cadherin-mediated adhesion, even in the absence of an unequivocal identification of the actual binding site and mode.
For the DETACH project, we set out to determine the X-ray crystallographic structures of human E-cadherin in complex with histamine, polycyclic aromatic hydrocarbons (PAHs) or other small aromatic compounds that might be present in the environment and be epidemiologically linked to an increased mortality or morbidity in exposed populations. These crystal structures should reveal how compounds that are structurally and chemically similar to the tryptophan side-chain that is used by the cadherins in their dimerization mechanism can bind into or in the vicinity of the E-cadherin hydrophobic pocket or cause structural changes that prevent E-cadherin dimerization, thus causing disruption in the integrity of the epithelial tissue. In the literature, several examples of either detrimental or beneficial inhibition of cadherin-mediated adhesion by small molecules are reported: histamine, an agonist released during inflammation and allergic response, modifies the permeability of human airway epithelia by interrupting the normal cadherin activity; the dietary phytochemical indol-3-carbinole is stimulating a growing interest as chemopreventive agent in several type of cancers, where it has been demonstrated to suppress both tumor cell growth and metastatic spread by interfering with E-cadherin mediated cell adhesion. A cyclic peptide derived from E-cadherin sequence (ADH-1 or Exeherin©) is currently in phase I clinical trials as anticancer drug. However, the total lack of structural details regarding these interactions makes it difficult to rationally design novel and more potent cadherin inhibitors to be screened as pharmacological agents. We tested all these compounds, and many others, in co-crystallization experiments but without success. This is consistent with the complete lack of any structural data regarding cadherin-small molecule complexes thus far available in the literature and by similar reported experiences by several other groups that have been working along the same lines. As already pointed out, the difficulty in crystallizing a small molecule-cadherin inhibitor is due to the low affinity/high dynamicity of the cadherin intermediates along the complex cadherin dimerization trajectory. However, it is widely believed that the quest for the first cadherin-small molecule complex will eventually result in the identification of a lead compound that can bind specifically into the cadherin binding pocket or in any other site where it can interfere with and modulate the cadherin dimerization mechanism. Such studies will pave the way to the design of potential ant-cancer and anti-inflammation agents.
We further set out to determine the X-ray crystallographic structures of E-cadherin bound to heavy metals. Cadherins are calcium dependent molecules. Three calcium ions are present at the interface between each cadherin extracellular domain. In vitro studies have shown that removal of calcium renders the cadherins inactive and prone to degradation. We hypothesize that replacement of Ca2+ with other bivalent cations such as Hg2+, Cu2+, Pb2+, Zn2+, Ni2+, Cd2+ and others, which are known toxic pollutants, might cause conformational changes in the structure of E-cadherin and interfere with the proper mechanism of homophilic binding, thus disrupting the integrity of the epithelial tissue and contributing to the permeability of the epithelial tissue, the body’s first line of defense. We intended to determine the crystal structure of metal-substituted human E-cadherin in order to analyze metal-induced structural changes that may affect cadherin dimerization. We did many experiments in which we replaced the natural Ca ions with other bivalent cations. As we always observed protein aggregation and precipitation, we clearly could not determine the crystal striucture of any metal-substituted E-cadherin fragment. However, our tests indirectly suggest that Ca displacement and substitution is detrimental to the activity of the protein and impairs proper cadherin folding.
Finally, we wanted to characterize the affinity and calculate the binding constant between E-cadherin and the small molecules that will appear to successfully compete with Trp2 for binding into the cadherin’s hydrophobic pocket using the Surface Plasmon Resonance (SPR) technique. In collaboration with Dr. L. Belvisi at the Universita’ Statale di Milano, and Dr. A. Tomassetti at the Istituto Nazionale dei Tumori, we developed a small library of peptidomimetics based on the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 (DWVI) of the N- and E-cadherin N-terminal “adhesive arm”. To our knowledge, this work represents the first attempt to rationally design small molecules targeting the strand dimer interfaces identified by crystallographic structures. Our compounds were tested in biochemical and cell adhesion assays for their ability to inhibit both N- and E-cadherin homophilic interactions relative to ADH-1. About 30 peptidomimetics of general formula NH3+-Asp-scaffold-Ile-NHCH3 were built in silico by replacing the central dipeptide Trp2-Val3 unit of the DWVI adhesive motif with several scaffolds developed in our and other laboratories. To evaluate the ability of each of the compounds of our virtual library to reproduce the DWVI key interactions observed in the X-ray dimer structures and during Molecular Dynamics simulations, we built a model of the EC1 fragment of E- and N-cadherin starting from the corresponding X-ray structures and set up a docking protocol using the Glide V5.7 software. Docking results were sorted according to the Glide score Based on this analysis, three ligands were selected for synthesis and biological assays. The three ligands were successfully synthesized using conventional high-yielding synthetic pathways. The three compounds were tested by ELISA for their ability to inhibit calcium-dependent cadherin binding using the N-cadherin-expressing epithelial ovarian cancer (EOC) cell line SKOV3 and N-cadherin-Fc chimeric protein. Two of these ligands at 2 mM concentration inhibited N-cadherin homophilic binding by 78% and 84%, respectively, and 50% and 65% at 1 mM concentration. Conversely, at 2 mM concentration ADH-1 and the third ligand provided about 50% inhibition of N-cadherin-Fc/cells interactions and appeared nearly ineffective at 1mM concentration. The compounds were then evaluated for their ability to inhibit EOC cells adhesion by observing the formation of cell monolayers in the presence of each ligand at two different concentrations (2 and 1 mM). As with ADH-1, at 2 mM concentration all compounds were able to inhibit the formation of cell monolayers of N-cadherin-expressing cells. Notably, the first two ligands were also active at 1 mM concentration, and the third was able to inhibit cell–cell aggregation of N-cadherin-expressing cells in suspension. When tested on the E-cadherin-expressing EOC cell line OAW42, all compounds were found to be less efficient in inhibiting both E-cadherin homophilic interactions and the formation of cell monolayers, with significant effects observed only at 2mM concentration. In particular, by ELISA, at the concentration of 2 mM two of the compounds gave about 50% inhibition of E-cadherin-Fc binding to the cells while ADH-1 showed 30% inhibition, indicating a slightly better efficacy compared to ADH-1 in inhibiting also E-cadherin homophilic interactions. The other compound was also able to inhibit the formation of cell monolayers at 1mM concentration. Overall, the first two compounds resulted to be effective in inhibiting N-cadherin homophilic and, to some extent, also E-cadherin homophilic adhesion. We also set out to evaluate by Surface Plasmon Resonance (SPR) analysis the ability of the three compounds to specifically inhibit the N-cadherin homo-dimerization in a cell-free experiment using the N-cadherin-Fc recombinant protein. At 10 μM concentrations, two of these peptidomimetcs provided 98% and 55% inhibition of N-cadherin homophilic binding, respectively, while ADH-1 showed only 28% inhibition. These data demonstrate the ability of both compounds to specifically bind to N-cadherin even in the μM range. The combination of our computational investigation with the results of SPR assays has started to shed light on the ligand structural requirements for binding to N-cadherin. In conclusion, two of our peptidomimetics inhibit the N-cadherin-mediated adhesion process in EOC cells with somewhat improved efficacy compared to the ADH-1 cyclic peptide, which is being investigated in phase I clinical trials as an N-cadhering antagonist in various tumors, including EOCs. Thus, the small molecules generated in the present study are likely to represent new leads for the development of a novel class of modulators of cadherin-mediated adhesion. Such compounds may play an important role in the investigation of cellular processes, and in the design of novel diagnostic and therapeutic approaches against tumors, especially EOCs.