Self-assembly of an anion receptor with metal-dependent kinase inhibition and potent *in vitro* anti-cancer properties

Anions play crucial roles in various cellular processes and cancer development. Despite this, anion receptors have received limited attention as potential anticancer therapeutics. This study investigates the potential of self-assembling trimetallic cryptands as novel anti-cancer agents. The research question focuses on whether these self-assembling structures, with their ability to bind and interact with anions, can selectively target and inhibit cancer cell growth. The context lies in the growing need for new cancer therapies, especially those that overcome drug resistance. The purpose is to synthesize, characterize, and evaluate the anticancer activity of a novel class of self-assembling trimetallic cryptands. The importance of the study stems from the potential to develop highly selective and effective anti-cancer drugs that minimize side effects on healthy tissues. The background explores the field of supramolecular chemistry, specifically anion binding and self-assembly, and how this relates to biological systems and cancer. The study’s innovation lies in utilizing the self-assembly process, coupled with metal-dependent reactivity and anion encapsulation, to design and tailor anticancer properties. Previous work in the field has established the potential of metallo-supramolecular complexes as anticancer agents, including those containing ruthenium, palladium and other transition metals. However, the design principles employed in this study, specifically focused on anion-binding and metal-dependent regulation of activity, offer a new approach for developing anticancer agents. This research aims to expand on this prior work by investigating a new class of metal complexes with enhanced selectivity and activity, highlighting the potential for targeted cancer therapy.

The paper extensively reviews the literature on supramolecular chemistry, specifically focusing on anion binding and self-assembly. It highlights the lack of attention given to anion receptors as anti-cancer therapeutics, despite the significance of anions in cellular processes and cancer development. The review covers previous research on metallo-supramolecular assemblies and their biomedical applications, including the exploration of metal-containing helicates and cages as potential anticancer agents. It references studies that have demonstrated the activity of other metallo-supramolecular complexes against cancer cells, some of which involve DNA binding. The paper acknowledges the success of platinum-based drugs in cancer treatment, while also pointing out their limitations, such as significant side effects and the emergence of drug resistance. The literature review establishes a strong rationale for exploring self-assembling anion receptors as a promising strategy for developing novel, more selective, and effective anti-cancer therapies. The review concludes by setting the current study in the context of the existing knowledge, highlighting its unique contribution through the combination of self-assembly, anion binding, and metal-dependent reactivity.

The study involved the synthesis of a ligand (L) and its subsequent self-assembly with various metal ions (Cu²⁺, Zn²⁺, Mn²⁺) and anions (PO₄³⁻, SO₄²⁻, PhOPO₃²⁻) to form different complexes. Detailed synthetic procedures, including reaction conditions and characterization techniques (¹H, ¹³C NMR, ESI-MS, elemental analysis) are provided in the supplementary information. Single-crystal X-ray crystallography was employed to determine the structures of several complexes, revealing the encapsulation of anions within the trimetallic cryptand structures. The metal-dependent reactivity of the complexes towards different anions, particularly phenyl phosphate, was investigated using ESI-MS and ¹H/³¹P NMR spectroscopy. These experiments demonstrated that the Zn²⁺ and Mn²⁺ complexes hydrolyzed phenyl phosphate to phosphate, while the Cu²⁺ complex bound to phenyl phosphate without hydrolysis. The phosphatase activity of the Zn²⁺ complex was further investigated using ³¹P NMR spectroscopy with various substrates, including phenyl phosphate and phosphorylated amino acids (serine, threonine, tyrosine). The rate of hydrolysis was found to be substrate-dependent, with differences attributed to both steric and electronic effects. The cytotoxic activity of the complexes against a panel of human cancer and non-cancer cell lines was assessed using the MTT assay. The IC₅₀ values were determined, and selectivity indices were calculated to evaluate the preferential activity against cancer cells. A cell-free kinase screen (using 140 purified recombinant human kinases) was conducted to examine the effects of the Zn²⁺ and Cu²⁺ complexes on kinase activity. ATP and substrate competition assays were performed to determine the mechanism of kinase inhibition. Finally, western blotting was employed to analyze the effects of the complexes on the phosphorylation levels of key regulatory amino acids in AMPK and Src kinases, and to assess the cellular uptake of the Zn²⁺ complex, autophagy, and ATP levels.

The study found that self-assembling trimetallic cryptands, particularly those containing copper (Cu²⁺) and zinc (Zn²⁺), exhibit potent and selective anticancer activity in vitro. Both [L₂Cu₃]⁶⁺ and [L₂Zn₃]⁶⁺ showed significant toxicity against various cancer cell lines (HT-29, DLD-1, HCT116, BxPC3, A549, H460), with IC₅₀ values mostly in the sub-µM range. Remarkably, [L₂Zn₃]⁶⁺ demonstrated selectivity indices exceeding 2000-fold against certain cancer cells compared to healthy cells (HCT116 p53⁻/⁻ vs. ARPE-19, MCF10A). The activity was modulated by the presence of different anions. The Zn²⁺ complex displayed phosphatase activity, hydrolyzing phosphate esters including phosphorylated amino acids (serine, tyrosine faster than threonine), while the Cu²⁺ complex bound to, but did not hydrolyze, phenyl phosphate. Both [L₂Zn₃]⁶⁺ and [L₂Cu₃]⁶⁺ showed differential and selective inhibition of multiple kinases in a cell-free screen, indicating non-competitive allosteric mechanisms. Western blotting revealed that [L₂Zn₃]⁶⁺ decreased phosphorylation levels of T172 in AMPKα, and S108 in AMPKβ, consistent with kinase inhibition. In contrast, [L₂Zn₃]⁶⁺ increased Y416 phosphorylation in Src, suggesting activation. The Zn²⁺ and Mn²⁺ complexes induced autophagy in cancer cells, correlating with dose-dependent ATP depletion. [L₂Zn₃]⁶⁺ also impaired glycolysis and mitochondrial respiration in HCT116 cancer cells. Longer treatment with [L₂Zn₃]⁶⁺ increased cellular levels of T172P AMPKα, attributed to ATP depletion. Both complexes increased p53 levels selectively in HCT116 cancer cells. The study demonstrates that the complexes can be modulated by choosing different metals or encapsulating different anions to alter their potency and selectivity, providing a versatile platform for drug discovery.

The findings demonstrate that self-assembling trimetallic cryptands can act as potent and selective anti-cancer agents, addressing the research question by showing effective cancer cell targeting. The significant selectivity observed, especially for the Zn²⁺ complex, highlights the potential for minimizing side effects compared to existing platinum-based therapies. The metal-dependent differences in reactivity (phosphatase activity for Zn²⁺/Mn²⁺ versus binding for Cu²⁺) and the modulation of activity by anion encapsulation emphasize the versatility of this self-assembly platform. The non-competitive mechanism of kinase inhibition suggests a potentially novel mode of action compared to most clinically used kinase inhibitors. The observed autophagy induction and ATP depletion suggest a complex interplay between the complexes’ effects on kinase activity and cellular energy metabolism. While the study focused on in vitro results, these promising findings warrant further investigation into in vivo efficacy and mechanisms. Future studies should explore the role of specific phospho-sites on proteins, particularly phospho-serine/threonine proline motifs, targeted by [L₂Zn₃]⁶⁺. Additionally, investigation into the precise mechanisms of kinase activation and the potential contributions of other mechanisms to the selective anti-cancer activity are crucial.

This study successfully synthesized and characterized self-assembling trimetallic cryptands with potent and selective anti-cancer properties. The Zn²⁺ and Cu²⁺ complexes showed remarkable activity and selectivity towards various cancer cell lines, achieved through unique mechanisms involving kinase modulation and potential ATP depletion. The ability to modulate activity and selectivity by choosing the metal and/or encapsulating specific anions offers a significant advantage for future drug development. Further research should focus on in vivo studies, detailed mechanistic investigations and exploration of additional therapeutic applications.

The study primarily focused on in vitro experiments, limiting the direct translation of findings to in vivo settings. The precise mechanisms by which the complexes selectively target cancer cells, particularly concerning kinase activation, warrant further investigation. The study used a limited number of cancer cell lines and healthy cell types, and further studies with a broader panel of cell types will strengthen the conclusions. The effects on other cellular pathways, besides those discussed, might contribute to the overall anticancer activity. It would also be advantageous to investigate the long-term effects of these compounds in cellular models. Finally, further studies are required to assess potential toxicity and side effects in vivo.